High Surface-Area Lyophilized Compositions Comprising Arsenic For Oral Administration In Patients

ABSTRACT

The present invention relates to treating malignancies such as tumors or cancers by orally administering lyophilized compositions comprising arsenic to a subject in such need. Malignancies include various hematological malignancies, such as acute myeloid leukemia (AML) including acute promyelocytic leukemia (APL), myelodysplastic syndrome (MDS), multiple myeloma (MM) and lymphomas and solid tumors including glioblastoma multiforme and breast cancer. This invention relates to a novel formulation comprising a lyophilized compositions comprising arsenic. The present invention also relates to a method for lyophilizing the arsenic trioxide, preparing the oral formulation comprising lyophilized compositions comprising arsenic, and a method for treating a subject with malignancies using the oral formulation.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a continuation of U.S. application Ser. No.16/852,758, filed Apr. 20, 2020, which is a continuation of U.S.application Ser. No. 16/299,214, filed on Mar. 12, 2019, now U.S. Pat.No. 10,653,628, which is a continuation of U.S. application Ser. No.16/148,193, filed Oct. 1, 2018, now U.S. Pat. No. 10,272,045, which is adivisional application of co-pending U.S. application Ser. No.15/012,355, filed on Feb. 1, 2016, now U.S. Pat. 10,111,836 which claimsthe benefit of U.S. Provisional Application No. 62/110,574, filed Feb.1, 2015, and U.S. Provisional Application No. 62/142,709, filed Apr. 3,2015, each of which is incorporated by reference herein in theirentirety.

TECHNICAL FIELD

The present invention relates to treating malignancies such as tumors orcancers by administering lyophilized compositions comprising arsenic toa subject. Malignancies include various hematological malignancies, suchas acute myeloid leukemia (AML) including acute promyelocytic leukemia(APL), myelodysplastic syndrome (MDS), multiple myeloma (MM), andlymphomas; and solid tumors including glioblastoma multiforme, andbreast cancer.

Conventional arsenic treatment has shown great promise in the treatmentof several cancers, but requires daily intravenous (IV) administration.In contrast, the oral formulation of the present invention provides asystemic bioavailability comparable to that of intravenous (IV)administration of arsenic trioxide that is being currently practiced. Italso exhibits the shelf life of more than three (3) months and providesa much more convenient, less risky, and less expensive method ofadministering arsenic trioxide than that provided by the intravenousadministration methods. The present invention also relates to method forpreparing the lyophilized composition comprising arsenic, method forpreparing the oral formulation of the present invention, method fororally administering the formulation to a subject, and a method fortreating a subject with malignancies, for example, hematologicalmalignancies, using the oral formulation.

BACKGROUND Hematological Malignancies

Hematological malignancies are cancers of the body's blood-formingsystems and immune systems. Hematological malignancies include, forexample, leukemia, lymphoma (both Hodgkin's disease and non-Hodgkin'slymphoma), and myeloma. The abnormal cell growth interferes with thebody's production of healthy blood cells, thus making the body unable toprotect itself against infections.

New cases of hematological malignancies account for about 9 percent ofcancer cases diagnosed in the United States, and about 59,200 personsare killed by the diseases each year. Many of these diseases occur inchildren.

Leukemia

Leukemia is a cancer of the bone marrow and blood. It is characterizedby the uncontrolled growth of blood cells. About 30,000 new cases ofleukemia are reported in the United States each year. Most cases occurin older adults, though leukemia is the most common type of childhoodcancer.

Leukemia is either acute or chronic. In acute leukemia, the abnormalblood cells are blasts that remain very immature and cannot carry outtheir normal functions. The number of blasts increases rapidly, and thedisease worsens rapidly. In chronic leukemia, some blast cells arepresent, but in general, these cells are more mature and can carry outsome of their normal functions. Also, the number of blasts increasesless rapidly than in acute leukemia. As a result, chronic leukemiaworsens gradually.

Leukemia can arise in either of the two main types of white bloodcells—lymphoid cells (lymphocytic leukemia) or myeloid cells (myeloid ormyelogenous leukemia). Common types of leukemia include acutelymphocytic leukemia (ALL); acute myeloid leukemia (AML) (sometimescalled acute nonlymphocytic leukemia (ANLL)) such as myeloblastic,promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemiasacute neutrophilic leukemia and myelodysplastic syndrome (MDS); chroniclymphocytic leukemia (CLL); chronic myeloid (granulocytic) leukemia(CML); chronic myelomonocytic leukemia (CMML); hairy cell leukemia; andpolycythemia vera and myeloproliferative neoplasms includingmyelofibrosis polycythemia vera and essential thrombocythemia.

Lymphoma

There are two main types of lymphoma—Hodgkin's disease and non-Hodgkin'slymphoma. Hodgkin's disease, also known as Hodgkin's lymphoma, is aspecial form of lymphoma in which a particular cell known as the ReedSternberg (R-S) cell occurs. This cell is not usually found in otherlymphomas.

The cause for Hodgkin's disease is unknown. Hodgkin's disease, likeother cancers, is not infectious and cannot be passed onto other people.It is not inherited. The first symptom of Hodgkin's disease is usually apainless swelling in the neck, armpits or groin. Other symptoms mayinclude night sweats or unexplained fever, weight loss and tiredness,cough or breathlessness, and persistent itch all over the body.

There are about 20 different types of non-Hodgkin's lymphoma.Non-Hodgkin's lymphomas are categorized according to their appearanceunder the microscope and the cell type (B-cell or T-cell). Risk factorsinclude old age, female, weakened immune system, human T-lymphotropicvirus type 1 (HTLV-1) and Epstein-Barr virus infection, and exposure tochemicals such as pesticides, solvents, and fertilizers.

Myeloma

Myeloma is a malignant tumor composed of plasma cells of the typenormally found in the bone marrow. Myeloma cells tend to collect in thebone marrow and in the hard, outer part of bones. Sometimes they collectin only one bone and form a single mass, or tumor, called aplasmacytoma. In most cases, however, the myeloma cells collect in manybones, often forming many tumors and causing other problems. When thishappens, the disease is called multiple myeloma such as, but not limitedto, giant cell myeloma, indolent myeloma, localized myeloma, multiplemyeloma, plasma cell myeloma, sclerosing myeloma, solitary myeloma,smoldering multiple myeloma, nonsecretary myeloma, osteoscleroticmyeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullaryplasmacytoma.

Myelodysplastic Syndromes

Myelodysplastic syndromes are disorders in which the bone marrowproduces ineffective and abnormal looking cells on one or more types(white blood cells, red blood cells or platelets). The majority ofpatients are men over sixty. Secondary myelodysplastic syndromes areseen following the use of chemotherapy and irradiation.

Signs and symptoms depend on the types of cells that are affected.Abnormal white cells make people more susceptible to infections;abnormal platelets make people more susceptible to bruising andspontaneous hemorrhages; and abnormal red blood cells causes anemia andfatigue.

While chemotherapy and radiation are useful in the treatment ofhematological malignancies, there is a continued need to find bettertreatment modalities and approaches to manage the disease that are moreeffective and less toxic, especially when clinical oncologists aregiving increased attention to the quality of life of cancer patients.The present invention provides an alternative approach to hematologicalmalignancies therapy and management of the disease by using an oralcomposition comprising arsenic trioxide.

Arsenic

Arsenic has been used medicinally for over 2,000 years. In the 18thcentury, a solution of arsenic trioxide (empirical formula As₂O₃) in 1%w/v potassium bicarbonate (Fowler's solution) was developed to treat avariety of infectious and malignant diseases. Its efficacy insuppressing white cells was first described in 1878 (Kwong Y. L. et al.Delicious poison: arsenic trioxide for the treatment of leukemia, Blood1997; 89: 3487-8). Arsenic trioxide was therefore used to treat chronicmyelogenous leukemia, until more potent cytotoxic drugs superseded it inthe 1940s. However, there was a resurgence of interest in such therapy,when arsenic trioxide was found to induce apoptosis and differentiationin acute promyelocytic leukemia (APL) cells (Chen G. Q. et al. Use ofarsenic trioxide (As₂O₃) in the treatment of acute promyelocyticleukemia (APL): I. As₂O₃ exerts dose-dependent dual effect on APL cellsin vitro and in vivo, Blood 1997; 89: 3345-53; Soignet S. L. et al.United States multicenter study of arsenic trioxide in relapsed acutepromyelocytic leukemia. J Clin Oncol. 2001; 19: 3852-60). The clinicalimplications of these in vitro observations have since been verified, asarsenic trioxide induces remissions in over 90% of such patients (ShenZ. X. et al. Use of arsenic trioxide (As₂O₃) in the treatment of acutepromyelocytic leukemia (APL): II. Clinical efficacy and pharmacokineticsin relapsed patients, Blood 1997; 89: 3354-60; Soignet S. L. et al.Complete remission after treatment of acute promyelocytic leukemia witharsenic trioxide, N Engl J Med 1998; 339: 1341-8; Niu C. et al., Studieson treatment of acute promyelocytic leukemia with arsenic trioxide:remission induction, follow-up and molecular monitoring in 11 newlydiagnosed and 47 relapsed acute promyelocytic leukemia patients, Blood1999; 94: 3315-24).

A typical course of arsenic trioxide involves daily intravenous (IV)administration for 4 to 8 weeks and the attendant inconvenience, risksand expense of maintaining suitable vascular access and prolongedhospitalization. Currently, there is no FDA-approved oral arsenictrioxide for clinical use. Fowler's solution is no longer detailed inmodem pharmacopoeias or listed in formularies (1941, Arsenum.Martindale, The Extra Pharmacopoeia 22: 209-15; British Pharmacopoeia.London: Her Majesty's Stationery Office, 1988; Appendix 1A, p Al2).Formulation comprising arsenic that could be orally administered couldtherefore offer distinct advantages.

The inventors of the present invention have arrived at a novelformulation that comprises a lyophilized composition comprising arsenicthat can be orally administered. The inventors have developed a methodfor making such novel formulation comprising lyophilized compositioncomprising arsenic. The lyophilized powder comprising arsenic isamenable to oral administration in patients, for example, via capsulesand tablets.

As₂O₃ powder is sparingly and extremely slowly soluble in cold water;even in boiling water it is only soluble in a 1:15 ratio (ArsenicTrioxide, In: Budavari S O'Neil M J (Eds), The Merck Index. Anencyclopedia of chemicals, drugs and biologicals. NJ: Merck & Co., Inc.11th Ed., Rahway, N.J., USA. 1989. Monograph 832, p 127). As a result,and because of other problems described previously, it has not beenformulated as an orally available composition.

SUMMARY

The present invention addresses this issue of insolubility and the lackof bioavailability of As₂O₃ and provides a lyophilized compositioncomprising arsenic that makes it bioavailable. The arsenic is introducedas As₂O₃ powder, which is then solubilized and lyophilized as describedinfra. More specifically, this invention relates to a lyophilizedcomposition comprising arsenic, in which the arsenic is present as oneor more salt of arsenic, and/or a solvate thereof, and/or As₂O₃, and/orone or more arsenic compounds.

Thus, as described herein, by “lyophilized composition comprisingarsenic” (LCCA) is meant a composition that comprises arsenic as one ormore of its salts, and/or a solvate thereof, and/or arsenic trioxideand/or any other compound comprising arsenic, said composition havingresulted from applying the method steps of the present inventiondescribing infra. The lyophilized composition comprising arsenic,alternatively, may be addressed herein as lyophilized arsenic trioxideor lyophilized As₂O₃ (LAT).

Clearly, the present invention envisions making this “lyophilizedcomposition comprising arsenic” through other methods.

Stated another way, the independent lyophilized composition comprisingarsenic is one aspect of the present invention. In one aspect, thisinvention also relates to the method of making the LCCA. In anotheraspect this invention also relates to an oral formulation comprising theLCCA. In yet another aspect, this invention relates to the method ofmaking such oral formulation. This invention also relates to apharmaceutical composition in a solid dosage form suitable for oraladministration, the composition comprising lyophilized compositioncomprising arsenic. This invention further relates to a pharmaceuticalcomposition in a solid dosage form suitable for oral administration, thecomposition comprising the LCCA, at least one bulking agent, and atleast one lubricant. In one aspect, this invention also relates to a kitcomprising the pharmaceutical composition comprising the LCCA. In yetanother aspect, this invention relates to a method of treatingmalignancies, such as hematological malignancies, in a patient in needthereof, comprising the step of administering to the patient atherapeutically effective amount of the pharmaceutical compositioncomprising the LCCA. Embodiments—Methods of Making LyophilizedCompositions Comprising Arsenic

This invention relates to a method for preparing lyophilized compositioncomprising arsenic (LCCA), said method comprising: (A) solubilizingAs₂O₃ powder in an aqueous medium to form an As₂O₃ solution; and (B)lyophilizing said As₂O₃ solution. In one embodiment, said solubilizingAs₂O₃ powder in an aqueous medium comprises: (I) adding an alkalizingagent to the As₂O₃ powder in a vessel, with or without stirring and withor without addition of water, to render a pH of about 12 or higher; (II)adding an acid to said vessel, with or without stirring and with orwithout addition of water, to adjust the pH to from about 7 to about 8;(III) optionally, adding a surfactant to said vessel, with or withoutstirring and with or without addition of water, and (IV) optionally,adding water to said vessel to generate an As₂O₃ solution with orwithout stirring. In another embodiment, for the methods described thusfar in this section, said alkalizing agent comprises sodium hydroxide(NaOH), sodium carbonate (Na₂CO₃), or a mixture thereof In yet anotherembodiment, the amount of said alkalizing agent added in the methodsdescribed above, is about 10% to about 100% the amount of the As₂O₃powder.

In one embodiment of the invention, for the methods described previouslyin this section, said acid comprises hydrochloric acid (HCl). In anotherembodiment, said HCl is about 6 M HCl. In a further embodiment, saidacid is added to said vessel to adjust the pH to about 7.2. In yetanother embodiment, for the methods described so far in this section,said surfactant comprises at least one of sodium lauryl sulfate; Tween80®; betacyclodextrin; poloxamer; tocopheryl polyethylene glycolsuccinate (TPGS). In another embodiment, said surfactant is added toabout 0.5% v/v to about 4.0% v/v, but does not exceed about 50% As₂O₃concentration.

In yet another embodiment of the present invention, for the methodsdescribed above in this section, said step of lyophilizing comprises:(A) freezing said As₂O₃ solution to generate a frozen As₂O₃ product; and(B) drying said As₂O₃ product to generate said lyophilized compositioncomprising arsenic. In another embodiment, for the methods described sofar, said freezing step comprises freezing said As₂O₃ solution at atemperature in the range of from about −50° C. to about 0° C. In anotherembodiment, said As₂O₃ solution is frozen at about −40° C. for at leastabout 6 hours. In yet another embodiment, said drying comprises at leastone of the following three conditions: (I) drying said As₂O₃ product atat least one temperature in the range of from about −40° C. to about 50°C., for the time in the range of from about 5 minutes to about 500 min;(II) drying said frozen As₂O₃ by progressively increasing temperaturefrom at least one first temperature in the range of from about −40° C.to about 50° C. to at least one second temperature in the range of fromabout −40° C. to about 50° C., for the time in the range of from about 5minutes to about 500 min, wherein said at least one second temperatureis higher than said at least one first temperature; and (III) applyingvacuum to said frozen As₂O₃ product in the range of from about 300millitorrs to about 1000 millitorrs, for the time in the range of fromabout 5 minutes to about 500 min.

In one embodiment of the invention, for the methods described so far inthis section, said drying step comprises heating said As₂O₃ product atabout −30° C. and about 800 millitorrs for about 60 minutes; heatingsaid As₂O₃ product from the previous step at −20° C. and 500 millitorrsfor about 120 minutes; heating said As₂O₃ product from the previous stepat about −5° C. and about 500 millitorrs for about 120 minutes; heatingsaid As₂O₃ product from the previous step at about 10° C. and about 500millitorrs for about 60 minutes; and heating said As₂O₃ product from theprevious step at about 25° C. and about 500 millitorrs for the time inthe range of from about 180 minutes to 300 minutes. In yet anotherembodiment, said drying step comprises heating said As₂O₃ product toabout −30° C. and about 800 millitorrs over about 60 minutes and holdingat about −30° C. and about 800 millitorrs for about 60 minutes; heatingsaid As₂O₃ product from the previous step to about −20° C. and about 500millitorrs over about 60 minutes and holding at about −20° C. and about500 millitorrs for about 120 minutes; heating said As₂O₃ product fromthe previous step to about −5° C. and about 500 millitorrs over about300 minutes and holding at about −5° C. and about 500 millitorrs forabout 120 minutes; heating said As₂O₃ product from the previous step toabout 10° C. and about 500 millitorrs over about 120 minutes and holdingat about 10° C. and about 500 millitorrs for about 60 minutes; andheating said As₂O₃ product from the previous step to about 25° C. andabout 500 millitorrs over about 60 minutes and holding at about 25° C.and about 500 millitorrs for the time in the range of from about 180minutes to 300 minutes.

Embodiments—Lyophilized Compositions Comprising Arsenic

This invention relates to a composition comprising lyophilizedcomposition comprising arsenic (LCCA). In one embodiment, suchcomposition further comprising at least one bulking agent, and at leastone lubricant. In another embodiment, said composition is prepared by amethod comprising: (A) solubilizing As₂O₃ powder in an aqueous medium toform an As₂O₃ solution; (B) lyophilizing said As₂O₃ solution to generatea lyopremix; (C) sifting said lyopremix to generate lyophilized As₂O₃powder; (D) optionally, adding at least one bulking agent to saidlyophilized As₂O₃ powder; (E) optionally, adding one or more lubricantsto said lyophilized As₂O₃ powder to generate said oral formulation ofAs₂O₃.

In one embodiment of the invention, for the compositions describedpreviously, said solubilizing As₂O₃ powder in an aqueous mediumcomprises: (I) adding an alkalizing agent to the As₂O₃ powder in avessel, with or without stirring and with or without addition of water,to render a pH of about 12 or higher; (II) adding an acid to saidvessel, with or without stirring and with or without addition of water,to adjust the pH to from about 7 to about 8; (III) optionally, adding asurfactant to said vessel, with or without stirring and with or withoutaddition of water, and (IV) optionally, adding water to said vessel togenerate an As₂O₃ solution with or without stirring. In anotherembodiment, for the compositions described thus far, said alkalizingagent comprises sodium hydroxide (NaOH), sodium carbonate (Na₂CO₃), or amixture thereof In yet another embodiment, the amount of said alkalizingagent added in the compositions described above, is about 10% to about100% the amount of the As₂O₃ powder.

In one embodiment of the invention, for the compositions describedpreviously, said acid comprises hydrochloric acid (HCl). In anotherembodiment, said HCl is about 6 M HCl. In a further embodiment, saidacid is added to said vessel to adjust the pH to about 7.2. In yetanother embodiment, for the compositions described so far, saidsurfactant comprises at least one of sodium lauryl sulfate; Tween 80®;betacyclodextrin; poloxamer; tocopheryl polyethylene glycol succinate(TPGS). In another embodiment, said surfactant is added to about 0.5%v/v to about 4.0% v/v, but does not exceed about 50% As₂O₃concentration.

In yet another embodiment of the present invention, for the compositionsdescribed above, said step of lyophilizing comprises: (A) freezing saidAs₂O₃ solution to generate a frozen As₂O₃ product; and (B) drying saidAs₂O₃ product to generate said lyophilized composition comprisingarsenic. In another embodiment, for the compositions described so far,said freezing step comprises freezing said As₂O₃ solution at atemperature in the range of from about −50° C. to about 0° C. In anotherembodiment, said As₂O₃ solution is frozen at about −40° C. for at leastabout 6 hours. In yet another embodiment, said drying comprises at leastone of the following three conditions: (I) drying said As₂O₃ product atat least one temperature in the range of from about −40° C. to about 50°C., for the time in the range of from about 5 minutes to about 500 min;(II) drying said frozen As₂O₃ by progressively increasing temperaturefrom at least one first temperature in the range of from about −40° C.to about 50° C. to at least one second temperature in the range of fromabout −40° C. to about 50° C., for the time in the range of from about 5minutes to about 500 min, wherein said at least one second temperatureis higher than said at least one first temperature; and (III) applyingvacuum to said frozen As₂O₃ product in the range of from about 300millitorrs to about 1000 millitorrs, for the time in the range of fromabout 5 minutes to about 500 min.

In one embodiment of the invention, for the compositions described sofar in this section, said drying step comprises heating said As₂O₃product at about −30° C. and about 800 millitorrs for about 60 minutes;heating said As₂O₃ product from the previous step at −20° C. and 500millitorrs for about 120 minutes; heating said As₂O₃ product from theprevious step at about −5° C. and about 500 millitorrs for about 120minutes; heating said As₂O₃ product from the previous step at about 10°C. and about 500 millitorrs for about 60 minutes; and heating said As₂O₃product from the previous step at about 25° C. and about 500 millitorrsfor the time in the range of from about 180 minutes to 300 minutes. Inyet another embodiment, said drying step comprises heating said As₂O₃product to about −30° C. and about 800 millitorrs over about 60 minutesand holding at about −30° C. and about 800 millitorrs for about 60minutes; heating said As₂O₃ product from the previous step to about −20°C. and about 500 millitorrs over about 60 minutes and holding at about−20° C. and about 500 millitorrs for about 120 minutes; heating saidAs₂O₃ product from the previous step to about −5° C. and about 500millitorrs over about 300 minutes and holding at about −5° C. and about500 millitorrs for about 120 minutes; heating said As₂O₃ product fromthe previous step to about 10° C. and about 500 millitorrs over about120 minutes and holding at about 10° C. and about 500 millitorrs forabout 60 minutes; and heating said As₂O₃ product from the previous stepto about 25° C. and about 500 millitorrs over about 60 minutes andholding at about 25° C. and about 500 millitorrs for the time in therange of from about 180 minutes to 300 minutes.

In another embodiment of the present invention, for the compositionsdescribed previously in this section, said bulking agent comprisesmannitol; and/or wherein said one or more lubricants comprises talcand/or magnesium stearate. In another embodiment, said composition is acontrolled-release, oral, solid, composition. In one embodiment, thecompositions described previously in this section are encapsulated in acapsule.

Embodiments—Methods for Preparing Oral Pharmaceutical FormulationComprising LCCA

This invention relates to a method for preparing an oral pharmaceuticalformulation comprising a lyophilized composition comprising arsenic orlyophilized arsenic trioxide (As₂O₃), said method comprising: (A)solubilizing As₂O₃ powder in an aqueous medium to form an As₂O₃solution; (B) lyophilizing said As₂O₃ solution to generate a lyopremix;(C) sifting said lyopremix to generate lyophilized As₂O₃ powder; (D)optionally, adding at least one bulking agent to said lyophilized As₂O₃powder; (E) optionally, adding one or more lubricants to saidlyophilized As₂O₃ powder to generate said oral formulation of As₂O₃.

This invention also relates to the method described previously, saidsolubilizing As₂O₃ powder in an aqueous medium comprises: (I) adding analkalizing agent to the As₂O₃ powder in a vessel, with or withoutstirring and with or without addition of water, to render a pH of about12 or higher; (II) adding an acid to said vessel, with or withoutstirring and with or without addition of water, to adjust the pH to fromabout 7 to about 8; (III) optionally, adding a surfactant to saidvessel, with or without stirring and with or without addition of water,and (IV) optionally, adding water to said vessel to generate an As₂O₃solution with or without stirring. In another embodiment, for themethods described thus far in this section, said alkalizing agentcomprises sodium hydroxide (NaOH), sodium carbonate (Na₂CO₃), or amixture thereof In yet another embodiment, the amount of said alkalizingagent added in the methods described above, is about 10% to about 100%the amount of the As₂O₃ powder.

In one embodiment of the invention, for the methods described previouslyin this section, said acid comprises hydrochloric acid (HCl). In anotherembodiment, said HCl is about 6 M HCl. In a further embodiment, saidacid is added to said vessel to adjust the pH to about 7.2. In yetanother embodiment, for the methods described so far in this section,said surfactant comprises at least one of sodium lauryl sulfate; Tween80®; betacyclodextrin; poloxamer; tocopheryl polyethylene glycolsuccinate (TPGS). In another embodiment, said surfactant is added toabout 0.5% v/v to about 4.0% v/v, but does not exceed about 50% As₂O₃concentration.

In yet another embodiment of the present invention, for the methodsdescribed above in this section, said step of lyophilizing comprises:(A) freezing said As₂O₃ solution to generate a frozen As₂O₃ product; and(B) drying said As₂O₃ product to generate said lyophilized compositioncomprising arsenic. In another embodiment, for the methods described sofar, said freezing step comprises freezing said As₂O₃ solution at atemperature in the range of from about −50° C. to about 0° C. In anotherembodiment, said As₂O₃ solution is frozen at about −40° C. for at leastabout 6 hours. In yet another embodiment, said drying comprises at leastone of the following three conditions: (I) drying said As₂O₃ product atat least one temperature in the range of from about −40° C. to about 50°C., for the time in the range of from about 5 minutes to about 500 min;(II) drying said frozen As₂O₃ by progressively increasing temperaturefrom at least one first temperature in the range of from about −40° C.to about 50° C. to at least one second temperature in the range of fromabout −40° C. to about 50° C., for the time in the range of from about 5minutes to about 500 min, wherein said at least one second temperatureis higher than said at least one first temperature; and (III) applyingvacuum to said frozen As₂O₃ product in the range of from about 300millitorrs to about 1000 millitorrs, for the time in the range of fromabout 5 minutes to about 500 min.

In one embodiment of the invention, for the methods described so far inthis section, said drying step comprises heating said As₂O₃ product atabout −30° C. and about 800 millitorrs for about 60 minutes; heatingsaid As₂O₃ product from the previous step at −20° C. and 500 millitorrsfor about 120 minutes; heating said As₂O₃ product from the previous stepat about −5° C. and about 500 millitorrs for about 120 minutes; heatingsaid As₂O₃ product from the previous step at about 10° C. and about 500millitorrs for about 60 minutes; and heating said As₂O₃ product from theprevious step at about 25° C. and about 500 millitorrs for the time inthe range of from about 180 minutes to 300 minutes. In yet anotherembodiment, said drying step comprises heating said As₂O₃ product toabout −30° C. and about 800 millitorrs over about 60 minutes and holdingat about −30° C. and about 800 millitorrs for about 60 minutes; heatingsaid As₂O₃ product from the previous step to about −20° C. and about 500millitorrs over about 60 minutes and holding at about −20° C. and about500 millitorrs for about 120 minutes; heating said As₂O₃ product fromthe previous step to about −5° C. and about 500 millitorrs over about300 minutes and holding at about −5° C. and about 500 millitorrs forabout 120 minutes; heating said As₂O₃ product from the previous step toabout 10° C. and about 500 millitorrs over about 120 minutes and holdingat about 10° C. and about 500 millitorrs for about 60 minutes; andheating said As₂O₃ product from the previous step to about 25° C. andabout 500 millitorrs over about 60 minutes and holding at about 25° C.and about 500 millitorrs for the time in the range of from about 180minutes to 300 minutes.

In another embodiment of the present invention, for the methodsdescribed previously in this section, said bulking agent comprisesmannitol; and/or wherein said one or more lubricants comprises talcand/or magnesium stearate. In another embodiment, methods describedpreviously further comprise the step of filling a capsule with said oralformulation.

Embodiments—Oral Pharmaceutical Formulation Comprising LCCA

This invention relates to a pharmaceutical composition in a solid dosageform suitable for oral administration, said composition comprisinglyophilized composition comprising arsenic. In one embodiment, suchpharmaceutical composition further comprises at least one bulking agent,and at least one lubricant. In yet another embodiment, thepharmaceutical compositions described so far in this section is preparedby a method comprising: (A) solubilizing As₂O₃ powder in an aqueousmedium to form an As₂O₃ solution; (B) lyophilizing said As₂O₃ solutionto generate a lyopremix; (C) sifting said lyopremix to generatelyophilized As₂O₃ powder; (D) optionally, adding at least one bulkingagent to said lyophilized As₂O₃ powder; (E) optionally, adding one ormore lubricants to said lyophilized As₂O₃ powder to generate said oralformulation of As₂O₃.

In one embodiment of the invention, for the pharmaceutical compositionsdescribed previously in this section, said solubilizing As₂O₃ powder inan aqueous medium comprises: (I) adding an alkalizing agent to the As₂O₃powder in a vessel, with or without stirring and with or withoutaddition of water, to render a pH of about 12 or higher; (II) adding anacid to said vessel, with or without stirring and with or withoutaddition of water, to adjust the pH to from about 7 to about 8; (III)optionally, adding a surfactant to said vessel, with or without stirringand with or without addition of water, and (IV) optionally, adding waterto said vessel to generate an As₂O₃ solution with or without stirring.In another embodiment, for the compositions described thus far, saidalkalizing agent comprises sodium hydroxide (NaOH), sodium carbonate(Na₂CO₃), or a mixture thereof In yet another embodiment, the amount ofsaid alkalizing agent added in the pharmaceutical compositions describedabove, is about 10% to about 100% the amount of the As₂O₃ powder.

In one embodiment of the invention, for the pharmaceutical compositionsdescribed previously, said acid comprises hydrochloric acid (HCl). Inanother embodiment, said HCl is about 6 M HCl. In a further embodiment,said acid is added to said vessel to adjust the pH to about 7.2. In yetanother embodiment, for the pharmaceutical compositions described sofar, said surfactant comprises at least one of sodium lauryl sulfate;Tween 80®; betacyclodextrin; poloxamer; tocopheryl polyethylene glycolsuccinate (TPGS). In another embodiment, said surfactant is added toabout 0.5% v/v to about 4.0% v/v, but does not exceed about 50% As₂O₃concentration.

In yet another embodiment of the present invention, for thepharmaceutical compositions described above, said step of lyophilizingcomprises: (A) freezing said As₂O₃ solution to generate a frozen As₂O₃product; and (B) drying said As₂O₃ product to generate said lyophilizedcomposition comprising arsenic. In another embodiment, for thecompositions described so far, said freezing step comprises freezingsaid As₂O₃ solution at a temperature in the range of from about −50° C.to about 0° C. In another embodiment, said As₂O₃ solution is frozen atabout −40° C. for at least about 6 hours. In yet another embodiment,said drying comprises at least one of the following three conditions:(I) drying said As₂O₃ product at at least one temperature in the rangeof from about −40° C. to about 50° C., for the time in the range of fromabout 5 minutes to about 500 min; (II) drying said frozen As₂O₃ byprogressively increasing temperature from at least one first temperaturein the range of from about −40° C. to about 50° C. to at least onesecond temperature in the range of from about −40° C. to about 50° C.,for the time in the range of from about 5 minutes to about 500 min,wherein said at least one second temperature is higher than said atleast one first temperature; and (III) applying vacuum to said frozenAs₂O₃ product in the range of from about 300 millitorrs to about 1000millitorrs, for the time in the range of from about 5 minutes to about500 min.

In one embodiment of the invention, for the pharmaceutical compositionsdescribed so far, said drying step comprises heating said As₂O₃ productat about −30° C. and about 800 millitorrs for about 60 minutes; heatingsaid As₂O₃ product from the previous step at −20° C. and 500 millitorrsfor about 120 minutes; heating said As₂O₃ product from the previous stepat about −5° C. and about 500 millitorrs for about 120 minutes; heatingsaid As₂O₃ product from the previous step at about 10° C. and about 500millitorrs for about 60 minutes; and heating said As₂O₃ product from theprevious step at about 25° C. and about 500 millitorrs for the time inthe range of from about 180 minutes to 300 minutes. In yet anotherembodiment, said drying step comprises heating said As₂O₃ product toabout −30° C. and about 800 millitorrs over about 60 minutes and holdingat about −30° C. and about 800 millitorrs for about 60 minutes; heatingsaid As₂O₃ product from the previous step to about −20° C. and about 500millitorrs over about 60 minutes and holding at about −20° C. and about500 millitorrs for about 120 minutes; heating said As₂O₃ product fromthe previous step to about −5° C. and about 500 millitorrs over about300 minutes and holding at about −5° C. and about 500 millitorrs forabout 120 minutes; heating said As₂O₃ product from the previous step toabout 10° C. and about 500 millitorrs over about 120 minutes and holdingat about 10° C. and about 500 millitorrs for about 60 minutes; andheating said As₂O₃ product from the previous step to about 25° C. andabout 500 millitorrs over about 60 minutes and holding at about 25° C.and about 500 millitorrs for the time in the range of from about 180minutes to 300 minutes.

In another embodiment of the present invention, for the pharmaceuticalcompositions described previously in this section, said bulking agentcomprises mannitol; and/or wherein said one or more lubricants comprisestalc and/or magnesium stearate. In another embodiment, saidpharmaceutical composition is a controlled-release, oral, solid,composition. In one embodiment, the pharmaceutical compositionsdescribed previously in this section are encapsulated in a capsule.

In another set of embodiments, the present invention also relates to acapsule comprising about 1 mg, about 5 mg, 10 mg, or about 20 mg of thepharmaceutical composition previously described in this section. Thisinvention further relates to a kit comprising the pharmaceuticalcomposition described in this section, and instructions for use of thesame.

Embodiments—Administration of Pharmaceutical Composition to Patients

This invention relates to a method of orally administering to a subject,a pharmaceutical composition comprising lyophilized compositioncomprising arsenic, comprising the steps of providing saidpharmaceutical composition, and orally administering said pharmaceuticalcomposition to said subject. This invention further relates to a methodof treating malignancies such as a cancer or a tumor in a patient inneed thereof, comprising the step of administering to the patient atherapeutically effective amount of the pharmaceutical compositioncomprising lyophilized composition comprising arsenic.

In yet another embodiment, this invention relates to the methodsdescribed previously in this section wherein said cancer is ahematological malignancy. In another embodiment, said hematologicalmalignancy is at least one of acute myeloid leukemia; acutenonlymphocytic leukemia; myeloblastic leukemia, promyelocytic leukemia;chronic myelomonocytic leukemia; monocytic leukemia; erythroleukemia;acute neutrophilic leukemia; myelodysplastic syndrome; acutepromyelocytic leukemia; chronic lymphocytic leukemia; chronic myeloidleukemia; hairy cell leukemia; myeloproliferative neoplasms; Hodgkin'slymphoma; non-Hodgkin's lymphoma; myeloma; giant cell myeloma; indolentmyeloma; localized myeloma; multiple myeloma; plasma cell myeloma;sclerosing myeloma; solitary myeloma; smoldering multiple myeloma;nonsecretary myeloma; osteosclerotic myeloma; plasma cell leukemia;solitary plasmacytoma; and extramedullary plasmacytoma.

In another embodiment, for the methods described thus far in thissection, said hematological malignancy is acute promyelocytic leukemia(APL). In one embodiment, said APL is newly diagnosed APL. In anotherembodiment, said APL is relapsed or refractory APL.

In one embodiment, in the methods of treatment described previously inthis section, said myeloproliferative neoplasm is one of myelofibrosispolycythemia vera and essential thrombocythemia.

In one embodiment of the present invention, for the methods described inthis section, said pharmaceutical composition is administered daily. Inanother embodiment, said wherein said pharmaceutical composition isadministered in a single dosage range of about 1 mg to about 50 mg. In afurther embodiment, said pharmaceutical composition is administered in asingle dosage range of about 0.1 mg/kg body weight to about 0.3 mg/kgbody weight.

In another embodiment, for the methods described previously in thissection, said patient was previously treated or is currently beingtreated with chemotherapy and/or radiation. In another embodiment, saidpatient is further administered one or more chemotherapeutic agents. Inone embodiment of this method, said chemotherapeutic agent isadministered before, after, or simultaneously with said pharmaceuticalcomposition.

Embodiments—Improvement in Physical/Chemical Characteristics of theFormulation Powder

This invention relates to a method for increasing the surface area oforiginal API powder comprising arsenic trioxide, from about 2× to about80× comprising the steps of: (A) solubilizing As₂O₃ powder in an aqueousmedium to form an As₂O₃ solution, comprising the steps of: (I) adding analkalizing agent to the As₂O₃ powder in a vessel, with or withoutstirring and with or without addition of water, to render a pH of about12 or higher; (II) adding an acid to said vessel, with or withoutstirring and with or without addition of water, to adjust the pH to fromabout 7 to about 8; (III) optionally, adding a surfactant to saidvessel, with or without stirring and with or without addition of water,and (IV) optionally, adding water to said vessel to generate an As₂O₃solution with or without stirring; and (B) lyophilizing said As₂O₃solution comprising the steps of: (V) freezing said As₂O₃ solution togenerate a frozen As₂O₃ product; and (VI) drying said As₂O₃ product togenerate said lyophilized composition comprising arsenic.

This invention also relates to a method for increasing the solubility ofarsenic trioxide powder in water or in alcohol by about 2× to about 30×,said method comprising the steps of: (A) solubilizing As₂O₃ powder in anaqueous medium to form an As₂O₃ solution, comprising the steps of: (I)adding an alkalizing agent to the As₂O₃ powder in a vessel, with orwithout stirring and with or without addition of water, to render a pHof about 12 or higher; (II) adding an acid to said vessel, with orwithout stirring and with or without addition of water, to adjust the pHto from about 7 to about 8; (III) optionally, adding a surfactant tosaid vessel, with or without stirring and with or without addition ofwater, and (IV) optionally, adding water to said vessel to generate anAs₂O₃ solution with or without stirring; and (B) lyophilizing said As₂O₃solution comprising the steps of: (V) freezing said As₂O₃ solution togenerate a frozen As₂O₃ product; and (VI) drying said As₂O₃ product togenerate said lyophilized composition comprising arsenic.

This invention also relates to a method for increasing the dissolutionof a pharmaceutical composition comprising arsenic, by at least fivetimes over the original API comprising arsenic trioxide, comprising thesteps of: (A) solubilizing As₂O₃ powder in an aqueous medium to form anAs₂O₃ solution, comprising the steps of: (I) adding an alkalizing agentto the As₂O₃ powder in a vessel, with or without stirring and with orwithout addition of water, to render a pH of about 12 or higher; (II)adding an acid to said vessel, with or without stirring and with orwithout addition of water, to adjust the pH to from about 7 to about 8;(III) optionally, adding a surfactant to said vessel, with or withoutstirring and with or without addition of water, and (IV) optionally,adding water to said vessel to generate an As₂O₃ solution with orwithout stirring; and (B) lyophilizing said As₂O₃ solution comprisingthe steps of: (V) freezing said As₂O₃ solution to generate a frozenAs₂O₃ product; and (VI) drying said As₂O₃ product to generate saidlyophilized composition comprising arsenic.

This invention also relates to a method for providing oralbioavailability of arsenic to a subject, wherein said oralbioavailability is in the range of from about 50% to about 100% of thatof the intravenous administration of a pharmaceutical compositioncomprising arsenic, said method comprising the steps of: (A)solubilizing As₂O₃ powder in an aqueous medium to form an As₂O₃solution, comprising the steps of: (I) adding an alkalizing agent to theAs₂O₃ powder in a vessel, with or without stirring and with or withoutaddition of water, to render a pH of about 12 or higher; (II) adding anacid to said vessel, with or without stirring and with or withoutaddition of water, to adjust the pH to from about 7 to about 8; (III)optionally, adding a surfactant to said vessel, with or without stirringand with or without addition of water, and (IV) optionally, adding waterto said vessel to generate an As₂O₃ solution with or without stirring;and (B) lyophilizing said As₂O₃ solution comprising the steps of: (V)freezing said As₂O₃ solution to generate a frozen As₂O₃ product; and(VI) drying said As₂O₃ product to generate said lyophilized compositioncomprising arsenic.

This invention also relates to the composition comprising lyophilizedcomposition comprising arsenic (LCCA), wherein said lyophilizedcomposition comprising arsenic comprises particles in the D(90) sizerange of from about 2 micron to 10 micron. In another embodiment, saidlyophilized composition comprising arsenic comprises particles having aD(90) size that is from about 10-times to about 50-times smaller thanthe particle size of the original API powder comprising arsenictrioxide. In yet another embodiment, said lyophilized compositioncomprising arsenic comprises particles having a BET surface area in therange of from about 0.5 m²/g to 5 m²/g. In yet another embodiment, saidlyophilized composition comprising arsenic comprises particles having aBET surface area that is from about 5 × to about 80 × more than thesurface area of the original API powder comprising arsenic trioxide. Inyet another embodiment, said lyophilized composition comprising arsenicis from about 2× to about 30× times more soluble in cold water or analcohol than the original API powder comprising arsenic trioxide. Inanother embodiment, said lyophilized composition comprising arsenic issoluble in cold water or alcohol in the range of from about 4 g/100 g toabout 60 g/100 g of the lyophilized composition to the cold water or thealcohol.

In one embodiment, the present invention relates to pharmaceuticalcompositions described in previous section, wherein the dissolution ofsaid pharmaceutical compositions as measured by ICP-OES is from about 5×to about 15× of that of the original API powder comprising arsenictrioxide.

This invention also relates to an oral dosage form in the form of acapsule which weighs 100 units and comprises: (I) lyophilizedcomposition comprising arsenic, at an amount that provides 10 units ofarsenic trioxide and 5 units sodium lauryl sulfate; (II) mannitol at anamount of 73 units; (III) talc at an amount of 1; and (IV) magnesiumstearate at an amount of 1 mg; which brings the total weight of thecomposition to 100 units. In one set of embodiment of the presentinvention, said capsule weighs 10 mg, 50 mg, 100 mg, or 200 mg.

For example, an oral dosage form in the form of a capsule which weighs10 mg and comprises:

-   -   (I) lyophilized composition comprising arsenic, at an amount        that provides 1 mg of arsenic trioxide and 0.5 mg sodium lauryl        sulfate;    -   (II) mannitol at an amount of 7.3 mg;    -   (III) talc at an amount of 0.1 mg; and    -   (IV) magnesium stearate at an amount of 0.1 mg    -   that brings the total weight of the composition to 10 mg.    -   Similarly, an oral dosage form in the form of a capsule which        weighs 50 mg and comprises:    -   (I) lyophilized composition comprising arsenic, at an amount        that provides 5 mg of arsenic trioxide and 2.5 mg sodium lauryl        sulfate;    -   (II) mannitol at an amount of 36.5 mg;    -   (III) talc at an amount of 0.5 mg; and    -   (IV) magnesium stearate at an amount of 0.5 mg    -   that brings the total weight of the composition to 50 mg.

In another example, an oral dosage form in the form of a capsule whichweighs 100 mg and comprises:

-   -   (I) lyophilized composition comprising arsenic, at an amount        that provides 10 mg of arsenic trioxide and 5 mg sodium lauryl        sulfate;    -   (II) mannitol at an amount of 73 mg;    -   (III) talc at an amount of 1 mg; and    -   (IV) magnesium stearate at an amount of 1 mg    -   that brings the total weight of the composition to 100 mg.

In yet another example, an oral dosage form in the form of a capsulewhich weighs 200 mg and comprises:

-   -   (I) lyophilized composition comprising arsenic, at an amount        that provides 20 mg of arsenic trioxide and 10 mg sodium lauryl        sulfate;    -   (II) mannitol at an amount of 146 mg;    -   (III) talc at an amount of 2 mg; and    -   (IV) magnesium stearate at an amount of 2 mg    -   that brings the total weight of the composition to 200 mg.

These, and other aspects of the present invention are described indetail infra.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows pharmacokinetic analysis in dogs of novel lyophilizedformulation comprising Arsenic and comparison with reference arsenictrioxide.

FIG. 2 shows the process flow diagram for lyophilization and capsulemaking.

FIG. 3 shows the X-Ray Diffractogram for the arsenic trioxide (API).

FIG. 4 shows the X-Ray Diffractogram for the Lyopremix of thelyophilized composition comprising arsenic.

FIG. 5 shows the X-Ray Diffractogram for the Final Blend (pharmaceuticalformulation) comprising the lyophilized composition comprising arsenic.

FIG. 6 . shows the X-Ray Diffractogram for the placebo.

FIG. 7 shows the SEM images of the arsenic trioxide API; the Lyopremix;and the Final Blend.

FIG. 8 . provides the BET Surface Area Plot for the arsenic trioxideAPI, which shows how the 1/[Q(Po/P−1)] depends on Relative PressureP/Po.

FIG. 9 provides the BET Surface Area Plot for the Lyopremix (lyophilizedcomposition comprising arsenic), which shows how the 1/[Q(Po/P−1)]depends on Relative Pressure P/Po.

DESCRIPTION OF THE INVENTION

This invention relates to a method for preparing lyophilized compositioncomprising arsenic (LCCA), said method comprising: (A) solubilizingAs₂O₃ powder in an aqueous medium to form an As₂O₃ solution; and (B)lyophilizing said As₂O₃ solution. In one embodiment, said solubilizingAs₂O₃ powder in an aqueous medium comprises: (I) adding an alkalizingagent to the As₂O₃ powder in a vessel, with or without stirring and withor without addition of water, to render a pH of about 12 or higher; (II)adding an acid to said vessel, with or without stirring and with orwithout addition of water, to adjust the pH to from about 7 to about 8;(III) optionally, adding a surfactant to said vessel, with or withoutstirring and with or without addition of water, and (IV) optionally,adding water to said vessel to generate an As₂O₃ solution with orwithout stirring.

In yet another embodiment of the present invention, for the methodsdescribed above in this section, said step of lyophilizing comprises:(A) freezing said As₂O₃ solution to generate a frozen As₂O₃ product; and(B) drying said As₂O₃ product to generate said lyophilized compositioncomprising arsenic. In another embodiment, for the methods described sofar, said freezing step comprises freezing said As₂O₃ solution at atemperature in the range of from about −50° C. to about 0° C. In anotherembodiment, said As₂O₃ solution is frozen at about −40° C. for at leastabout 6 hours.

This invention also relates to a composition comprising lyophilizedcomposition comprising arsenic (LCCA). In one embodiment, suchcomposition further comprising at least one bulking agent, and at leastone lubricant. In another embodiment, said composition is prepared by amethod comprising: (A) solubilizing As₂O₃ powder in an aqueous medium toform an As₂O₃ solution; (B) lyophilizing said As₂O₃ solution to generatea lyopremix; (C) sifting said lyopremix to generate lyophilized As₂O₃powder; (D) optionally, adding at least one bulking agent to saidlyophilized As₂O₃ powder; (E) optionally, adding one or more lubricantsto said lyophilized As₂O₃ powder to generate said oral formulation ofAs₂O₃.

In another embodiment of the present invention, for the compositionsdescribed previously in this section, said bulking agent comprisesmannitol; and/or wherein said one or more lubricants comprises talcand/or magnesium stearate. In another embodiment, said composition is acontrolled-release, oral, solid, composition. In one embodiment, thecompositions described previously in this section are encapsulated in acapsule.

This invention also relates to a method for preparing an oralpharmaceutical formulation comprising a lyophilized compositioncomprising arsenic or lyophilized arsenic trioxide (As₂O₃), said methodcomprising: (A) solubilizing As₂O₃ powder in an aqueous medium to forman As₂O₃ solution; (B) lyophilizing said As₂O₃ solution to generate alyopremix; (C) sifting said lyopremix to generate lyophilized As₂O₃powder; (D) optionally, adding at least one bulking agent to saidlyophilized As₂O₃ powder; (E) optionally, adding one or more lubricantsto said lyophilized As₂O₃ powder to generate said oral formulation ofAs₂O₃. This invention relates to a pharmaceutical composition in a soliddosage form suitable for oral administration, said compositioncomprising lyophilized composition comprising arsenic. In oneembodiment, such pharmaceutical composition further comprises at leastone bulking agent, and at least one lubricant. In yet anotherembodiment, the pharmaceutical compositions described so far in thissection is prepared by the method described herein. In anotherembodiment of the present invention, for the pharmaceutical compositionsdescribed previously in this section, said bulking agent comprisesmannitol; and/or wherein said one or more lubricants comprises talcand/or magnesium stearate. In another embodiment, said pharmaceuticalcomposition is a controlled-release, oral, solid, composition. In oneembodiment, the pharmaceutical compositions described previously in thissection are encapsulated in a capsule.

In another set of embodiments, the present invention also relates to acapsule comprising about 1 mg, about 5 mg, 10 mg, or about 20 mg of thepharmaceutical composition previously described in this section. Thisinvention further relates to a kit comprising the pharmaceuticalcomposition described in this section, and instructions for use of thesame.

This invention relates to a method of orally administering to a subject,a pharmaceutical composition comprising lyophilized compositioncomprising arsenic, comprising the steps of providing saidpharmaceutical composition, and orally administering said pharmaceuticalcomposition to said subject. This invention further relates to a methodof treating a cancer in a patient in need thereof, comprising the stepof administering to the patient a therapeutically effective amount ofthe pharmaceutical composition comprising lyophilized compositioncomprising arsenic.

This invention relates to a method for increasing the surface area oforiginal API powder comprising arsenic trioxide, from about 5× to about80 ×; for increasing the solubility of arsenic trioxide powder in wateror in alcohol by about 2× to about 30×; for increasing the dissolutionof a pharmaceutical composition comprising arsenic, by at least fivetimes over the original API; for providing oral bioavailability ofarsenic to a subject, wherein said oral bioavailability is in the rangeof from about 50% to about 100% of that of the intravenousadministration of a pharmaceutical composition comprising arsenic; andfor reducing the particles in the D(90) size range to from about 2micron to about 10 micron.

This invention also relates to an oral dosage form in the form of acapsule which weighs 100 units and comprises: (I) lyophilizedcomposition comprising arsenic, at an amount that provides 10 units ofarsenic trioxide and 5 units sodium lauryl sulfate; (II) mannitol at anamount of 73 units; (III) talc at an amount of 1; and (IV) magnesiumstearate at an amount of 1 mg; which brings the total weight of thecomposition to 100 units. In one set of embodiment of the presentinvention, said capsule weighs 10 mg, 50 mg, 100 mg, or 200 mg.

Methods of Use Use in Subjects with Hematological Malignancies

The present invention further provides methods of using the orallyadministered lyophilized composition comprising arsenic. In oneembodiment, the lyophilized composition comprising arsenic is used as amedicament for treatment of hematological malignancies (e.g., leukemia,Hodgkin's disease, non-Hodgkin's lymphoma, and myeloma). The methodscomprise administering an effective amount of the lyophilizedcomposition comprising arsenic to a subject in need. The lyophilizedcomposition comprising arsenic may be administered orally, in alyophilized form, or enterally through a feeding tube. As used herein,the term “an effective amount” means an amount sufficient to provide atherapeutic or healthful benefit in the context of hematologicalmalignancies.

In one embodiment, the lyophilized composition comprising arsenic canproduce a healthful benefit in a subject suffering from hematologicalmalignancies. Preferably, the subject is a human being. The subject inneed is one who is diagnosed with hematological malignancies, with orwithout metastasis, at any stage of the disease. As used herein, theterm “hematological malignancies” include but are not limited toleukemia, lymphoma, and myeloma. As used herein, the term “leukemia”includes but is not limited to acute lymphocytic leukemia (ALL); acutemyeloid leukemia (AML) (sometimes called acute nonlymphocytic leukemia(ANLL)) such as myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukemia leukemias and myelodysplastic syndrome; chroniclymphocytic leukemia (CLL); chronic myeloid (granulocytic) leukemia(CIVIL); chronic myelomonocytic leukemias (CMML); hairy cell leukemia;and polycythemia vera. As used herein, the term “lymphoma” includes butis not limited to Hodgkin's disease and non-Hodgkin's lymphoma. As usedherein, the term “myeloma” includes but is not limited to giant cellmyeloma, indolent myeloma, localized myeloma, multiple myeloma, plasmacell myeloma, sclerosing myeloma, solitary myeloma, smoldering multiplemyeloma, nonsecretary myeloma, osteosclerotic myeloma, plasma cellleukemia, solitary plasmacytoma, and extramedullary plasmacytoma.

The subject may be a patient who is receiving concurrently othertreatment modalities against the hematological malignancies. The subjectcan be a patient with hematological malignancies who had undergone aregimen of treatment (e.g., chemotherapy and/or radiation) and whosecancer is regressing. The subject may be a patient with hematologicalmalignancies who had undergone a regimen of treatment and who appears tobe clinically free of the hematological malignancies. The lyophilizedcomposition comprising arsenic of the invention can be orallyadministered adjunctively with any of the treatment modalities, such asbut not limited to chemotherapy and/or radiation. For example, thearsenic trioxide composition can be used in combination with one or morechemotherapeutic or immunotherapeutic agents, such as amsacrine (AMSA),busulfan (Myleran(R)), chlorambucil (Leukeran(R)), cladribine(2-chlorodeoxyadenosine; “2-CDA”; Leustatin(R)), cyclophosphamide(Cytoxan(R)), cytarabine (ara-C;Cytosar-U(R)), daunorubicin(Cerubidine(R)), doxorubicin (Adriamycin(R)), etoposide (VePesid(R)),fludarabine phosphate (Fludara(R)), hydroxyurea (Hydrea(R)), idarubicin(Idamycin(R)), L-asparaginase (Elspar(R)), methotrexate sodium plus6-mercaptopurine (6-MP; Purinethol(R)), mitoxantrone (Novantrone(R)),pentostatin (2-deoxycoformycin; “DCF”; Nipent(R)), prednisone, retinoicacid (ATRA), vincristine sulfate (Oncovin(R)), 6-thioguanine(Tabloid(R)), cyclosporin A, Taxol(R)), Cisplatin(R), Carboplatin(R),Doxil(R), Topotecan(R), Methotrexate(R), Bleomycin(R), andEpirubicin(R). The arsenic trioxide composition can also be used afterother regimen(s) of treatment is concluded.

The subject may be one who has not yet been diagnosed with hematologicalmalignancies but are predisposed to or at high risk of developinghematological malignancies as a result of genetic factors and/orenvironmental factors.

Depending on the subject, the therapeutic and healthful benefits rangefrom inhibiting or retarding the growth of the hematologicalmalignancies and/or the spread of the hematological malignancies toother parts of the body (i.e., metastasis), palliating the symptoms ofthe cancer, improving the probability of survival of the subject withthe cancer, prolonging the life expectancy of the subject, improving thequality of life of the subject, and/or reducing the probability ofrelapse after a successful course of treatment (e.g., chemotherapy,radiation). The symptoms associated with hematological malignanciesinclude but are not limited to a weakened immune system, infections,fevers, decrease in red blood cells and platelets, weakness, fatigue,loss of appetite, loss of weight, swollen or tender lymph nodes, liver,or spleen, easy bleeding or bruising, tiny red spots (called petechiae)under the skin, swollen or bleeding gums, sweating (especially atnight), bone or joint pain, headaches, vomiting, confusion, loss ofmuscle control, and seizures.

In particular, the invention provides a method for complete remission ofthe hematological malignancies in a subject, such as a human, comprisingadministering orally to the subject an arsenic trioxide composition ofthe invention. In other embodiments, the invention provides at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95% remission of thehematological malignancy. The invention also provide a method forprolonging the time of survival of a subject inflicted withhematological malignancies, preferably a human patient, comprisingadministering orally to the subject a lyophilized composition comprisingarsenic of the invention.

The effective dose will vary with the subject treated and the route ofadministration. The effective dose for the subject will also vary withthe condition to be treated and the severity of the condition to betreated. The dose, and perhaps the dose frequency, will also varyaccording to the age, body weight, and response of the individualsubject. In general, the total daily dose range of the lyophilizedcomposition comprising arsenic for a subject inflicted withhematological malignancies in mg is as follows: 1, 2, 3, 4, 5, 6, 7, 8,9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, and 50 mg per day. Depending upon the need of thepatient it can be higher, for example, 60, 70, 80, 90, and 100 mg/day,or any intermediate dosage numbers therebetween. The lyophilized premixcomprising arsenic composition is administered to the subject orally.

The length of time for a course of treatment should be at least 1 day,at least 2 days, at least 3 days, at least 4 days, at least 5 days, atleast 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, atleast 4 weeks, at least 5 weeks, at least 7 weeks, at least 10 weeks, atleast 13 weeks, at least 15 weeks, at least 20 weeks, at least 6 months,at least 1 year, or at least two years. It may be necessary to usedosages outside these ranges in some cases as will be apparent to thoseskilled in the art. In certain embodiments, the lyophilized compositioncomprising arsenic can be administered for a period of time until thesymptoms are under control, or when the disease has regressed partiallyor completely. Further, it is noted that the clinician or treatingphysician will know how and when to interrupt, adjust, or terminate useof the arsenic trioxide composition as a medication in conjunction withindividual patient response.

The effect of the lyophilized composition comprising arsenic of theinvention on development and progression of hematological malignanciescan be monitored by any methods known to one skilled in the art,including but not limited to measuring: a) changes in the size andmorphology of the tumor using imaging techniques such as a computedtomographic (CT) scan or a sonogram; and b) changes in levels ofbiological markers of risk for hematological malignancies.

In certain embodiments, toxicity and efficacy of the prophylactic and/ortherapeutic protocols of the instant invention can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Prophylactic and/or therapeutic agents that exhibit large therapeuticindices are preferred. While prophylactic and/or therapeutic agents thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

In other embodiments, the data obtained from the cell culture assays andanimal studies can be used in formulating a range of dosage of theprophylactic and/or therapeutic agents for use in humans. The dosage ofsuch agents lies preferably within a range of circulating concentrationsthat include the ED50 with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any agent used in the method of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the test compound that achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

The anti-cancer activity of the therapies used in accordance with thepresent invention also can be determined by using various experimentalanimal models for the study of cancer such as the acid mouse model ortransgenic mice. The following are some assays provided as examples andnot by limitation.

The lyophilized composition comprising arsenic of the present inventionis prepared as described below in the description and the experimentalsection. Arsenic or one or more of its chemical forms is the activeingredient, and can optionally contain a pharmaceutically acceptablecarrier or excipient, and/or other ingredients provided that theseingredients do not compromise (e.g., reduce) the efficacy lyophilizedcomposition comprising arsenic. Other ingredients that can beincorporated into lyophilized composition comprising arsenic of thepresent invention, may include, but are not limited to, herbs (includingtraditional Chinese medicine products), herbal extracts, vitamins, aminoacids, metal salts, metal chelates, coloring agents, flavor enhancers,preservatives, and the like.

Any dosage form may be employed for providing the subject with aneffective dosage of the oral composition. Dosage forms include tablets,capsules, dispersions, suspensions, solutions, and the like. In oneembodiment, compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,blisters, cachets, or tablets, each containing a predetermined amount ofactivated and conditioned yeast cells, as a powder or granules or as asolution or a suspension in an aqueous liquid, a non-aqueous liquid, anoil-in-water emulsion, or a water-in-oil liquid emulsion. In preferredembodiments, the oral composition is in the form of a lyophilizedpowdery or fluffy solid. In general, the compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired presentation. Suchproducts can be used as pharmaceuticals or dietary supplements,depending on the dosage and circumstances of its use.

The oral compositions of the present invention may additionally includebinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); binders or fillers (e.g., lactose,pentosan, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulfate). The tablets or capsules can be coated bymethods well known in the art.

In certain embodiments, lyophilized composition comprising arseniccomprises about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 21, 22, 23, 24, or 25 mg of theequivalent of arsenic trioxide per ml.

Generally, because of their ease of administration, tablets and capsulesrepresent the most advantageous oral dosage unit form, in which casesolid pharmaceutical carriers as described above are employed. In apreferred embodiment, the composition is a capsule. The capsules can beformulated by any commercially available methods. In certainembodiments, the composition is a capsule containing 0.25 mg, 0.5 mg, 1mg, 2 mg, 3, mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42mg, 43 mg, 24 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, or 50 mg of thelyophilized composition comprising arsenic in powder form.

Additional examples of anti-cancer agents that can be used in thevarious embodiments of the invention, including pharmaceuticalcompositions and dosage forms and kits of the invention, include, butare not limited to: acivicin; aclarubicin; acodazole hydrochloride;acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantroneacetate; aminoglutethimide; amsacrine; anastrozole; anthramycin;asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat;benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicinhydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin;enpromate; epipropidine; epirubicin hydrochloride; erbulozole;esorubicin hydrochloride; estramustine; estramustine phosphate sodium;etanidazole; etoposide; etoposide phosphate; etoprine; fadrozolehydrochloride; fazarabine; fenretinide; floxuridine; fludarabinephosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; ilmofosine; interleukin II (includingrecombinant interleukin II, or rIL2); interferon alfa-2a; interferonalfa-2b; interferon alfa-n1; interferon alfa-n3; interferon beta-Ia;interferon gamma-Ib; iproplatin; irinotecan hydrochloride; lanreotideacetate; letrozole; leuprolide acetate; liarozole hydrochloride;lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol;maytansine; mechlorethamine hydrochloride; megestrol acetate;melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide;mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper;mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole;nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium;tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride. Other anti-cancer drugs include, but are not limited to:20-epi-1,25 dihydroxyvitaminutes D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; 9-dihydrotaxol; dioxamycin; diphenylspiromustine; docetaxel; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflomithine; elemene; emitefur; epirubicin;epristeride; estramustine analogue; estrogen agonists; estrogenantagonists; etanidazole; etoposide phosphate; exemestane; fadrozole;fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; 4-ipomeanol; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; rasinhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred additional anti-cancer drugs are 5-fluorouraciland leucovorin. These two agents are particularly useful when used inmethods employing thalidomide and a topoisomerase inhibitor.

Formulations

In a preferred embodiment, this invention relates to a method forpreparing lyophilized composition comprising arsenic, alternativelyaddressed herein as lyophilized arsenic trioxide (As₂O₃), said methodcomprising:

-   -   a. solubilizing As₂O₃ powder in water in a vessel, said        solubilizing comprising, in the following order:        -   i. adding an alkalizing agent to said vessel to a pH of            about 12 or higher,        -   ii. adding an acid to said vessel to adjust the pH to about            7 to about 8,        -   iii. adding a surfactant to said vessel, and        -   iv. adding water to said vessel to generate an As₂O₃            solution; and    -   b. lyophilizing said As₂O₃ solution.

In one embodiment of the above method, the alkalizing agent comprisessodium hydroxide (NaOH) or sodium carbonate (Na₂CO₃). In anotherembodiment, the amount of said alkalizing agent added is about 10% toabout 100% the amount of the As₂O₃ powder. Stated another way, theamount of said alkalizing agent added is about 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95 96, 97, 98, 99, and 100% the amountof the As₂O₃ powder. In another embodiment, the amount of saidalkalizing agent added is any number within a range defined by andinclusive of any two numbers stated above.

In yet another embodiment, said acid comprises hydrochloric acid (HCl).In one embodiment, said HCl is about 6 M HCl. In one embodiment, theacid is added to said vessel to adjust the pH to about 7.2. In oneembodiment, the water is added to said vessel after step a)i), a)ii),a)iii), and/or a)iv).

In one embodiment of the invention, the surfactant comprises sodiumlauryl sulfate; Tween 80; betacyclodextrin; poloxamer; tocopherylpolyethylene glycol succinate (TPGS). In one embodiment of theinvention, the surfactant is added to about 0.5% v/v to about 4.0% v/v,but does not exceed about 50% As₂O₃ concentration.

In one embodiment of the invention, the step of lyophilizing comprisesfreezing said As₂O₃ solution to generate a frozen As₂O₃ product anddrying said frozen As₂O₃ product to generate said lyophilized As₂O₃ orthe lyophilized composition comprising arsenic. In another embodiment,the freezing step comprises freezing said As₂O₃ solution at about −40°C. In another embodiment, the As₂O₃ solution is frozen at about −40° C.for at least about 6 hours. In one embodiment, the drying step comprisesapplying heat and a vacuum to said As₂O₃ solid. For example, the dryingstep comprises heating said As₂O₃ product at about −30° C. and about 800millitorrs for about 60 minutes; heating said As₂O₃ product at −20° C.and 500 millitorrs for about 120 minutes; heating said As₂O₃ product atabout −5° C. and about 500 millitorrs for about 120 minutes; heatingsaid As₂O₃ product at about 10° C. and about 500 millitorrs for about 60minutes; heating said As₂O₃ product at about 25° C. and about 500millitorrs for about 180 minutes.

In another embodiment, said drying step comprises heating said As₂O₃product to about −30° C. and about 800 millitorrs over about 60 minutesand holding at about −30° C. and about 800 millitorrs for about 60minutes; heating said As₂O₃ product to about −20° C. and about 500millitorrs over about 60 minutes and holding at about −20° C. and about500 millitorrs for about 120 minutes; heating said As₂O₃ product toabout −5° C. and about 500 millitorrs over about 300 minutes and holdingat about −5° C. and about 500 millitorrs for about 120 minutes; heatingsaid As₂O₃ product to about 10° C. and about 500 millitorrs over about120 minutes and holding at about 10° C. and about 500 millitorrs forabout 60 minutes; and heating said As₂O₃ product to about 25° C. andabout 500 millitorrs over about 60 minutes and holding at about 25° C.and about 500 millitorrs for about 180 minutes.

In one embodiment, the drying step further comprises, following saidheating of said As₂O₃ product at about 25° C. and about 500 millitorrsfor about 180 minutes, heating said As₂O₃ product at about 25° C. andabout 500 millitorrs for about 120 minutes.

This invention also relates to a method for preparing an oralformulation comprising LCCA, said method comprising:

-   -   c. solubilizing As₂O₃ powder in water in a vessel, said        solubilizing comprising, in the following order:        -   i. adding an alkalizing agent to said vessel to a pH of            about 12 or higher,        -   ii. adding an acid to said vessel to adjust the pH to about            7 to about 8,        -   iii. adding a surfactant to said vessel, and        -   iv. and adding water to said vessel to generate an As₂O₃            solution; and    -   d. lyophilizing said As₂O₃ solution to generate a lyopremix;    -   e. sifting said lyopremix to generate the LCCA powder;    -   f. adding a bulking agent to said LCCA powder;    -   g. adding one or more lubricants to said LCCA powder to generate        said oral formulation comprising LCCA.

In one embodiment of the above method, the alkalizing agent comprisessodium hydroxide (NaOH) or sodium carbonate (Na₂CO₃). In anotherembodiment, the amount of said alkalizing agent added is about 10% toabout 100% the amount of the As₂O₃ powder. In yet another embodiment,said acid comprises hydrochloric acid (HCl). In one embodiment, said HClis about 6 M HCl. In one embodiment, the acid is added to said thevessel to adjust the pH to about 7.2. In one embodiment, the water isadded to said vessel after step c)i), c)ii), c)iii), and/or c)iv).

In one embodiment of the invention, the surfactant comprises sodiumlauryl sulfate; Tween 80; betacyclodextrin; poloxamer; tocopherylpolyethylene glycol succinate (TPGS). In one embodiment of theinvention, the surfactant is added to about 0.5% v/v to about 4.0% v/v,but does not exceed about 50% As₂O₃ concentration.

In one embodiment of the invention, the step of lyophilizing comprisesfreezing said As₂O₃ solution to generate a frozen As₂O₃ product anddrying said frozen As₂O₃ product to generate said lyophilized As₂O₃. Inanother embodiment, the freezing step comprises freezing said As₂O₃solution at about −40° C. In another embodiment, the As₂O₃ solution isfrozen at about −40° C. for at least about 6 hours. In one embodiment,the drying step comprises applying heat and a vacuum to said As₂O₃product solid. For example, the drying step comprises heating said As₂O₃product at about −30° C. and about 800 millitorrs for about 60 minutes;heating said As₂O₃ product at −20° C. and 500 millitorrs for about 120minutes; heating said As₂O₃ product at about −5° C. and about 500millitorrs for about 120 minutes; heating said As₂O₃ product at about10° C. and about 500 millitorrs for about 60 minutes; heating said As₂O₃product at about 25° C. and about 500 millitorrs for about 180 minutes.

In another embodiment, said drying step comprises heating said As₂O₃product to about −30° C. and about 800 millitorrs over about 60 minutesand holding at about −30° C. and about 800 millitorrs for about 60minutes; heating said As₂O₃ product to about −20° C. and about 500millitorrs over about 60 minutes and holding at about −20° C. and about500 millitorrs for about 120 minutes; heating said As₂O₃ product toabout −5° C. and about 500 millitorrs over about 300 minutes and holdingat about −5° C. and about 500 millitorrs for about 120 minutes; heatingsaid As₂O₃ product to about 10° C. and about 500 millitorrs over about120 minutes and holding at about 10° C. and about 500 millitorrs forabout 60 minutes; and heating said As₂O₃ product to about 25° C. andabout 500 millitorrs over about 60 minutes and holding at about 25° C.and about 500 millitorrs for about 180 minutes.

In one embodiment, the drying step further comprises, following saidheating of said As₂O₃ product at about 25° C. and about 500 millitorrsfor about 180 minutes, heating said As₂O₃ product at about 25° C. andabout 500 millitorrs for about 120 minutes.

In one embodiment of the above invention, the bulking agent comprisesmannitol. In another embodiment, the one or more lubricants comprisestalc and/or magnesium stearate.

In a further step, the above invention comprises filling a capsule withsaid oral formulation.

This invention also relates to a pharmaceutical composition in a soliddosage form suitable for oral administration, the composition comprisinglyophilized arsenic trioxide also known as lyophilized compositioncomprising arsenic, at least one bulking agent, and at least onelubricant.

In one embodiment, the pharmaceutical composition above is produced by amethod comprising:

-   -   h. solubilizing As₂O₃ powder in water in a vessel, said        solubilizing comprising, in the following order:        -   v. adding an alkalizing agent to said vessel to a pH of            about 12 or higher,        -   vi. adding an acid to said vessel to adjust the pH to about            7 to about 8,        -   vii. adding a surfactant to said vessel, and        -   viii. and adding water to said vessel to generate an As₂O₃            solution; and    -   i. lyophilizing said As₂O₃ solution to generate a lyopremix;    -   j. sifting said lyopremix to generate the LCCA powder;    -   k. adding a bulking agent to said LCCA powder;    -   l. adding one or more lubricants to said LCCA powder to generate        said oral formulation of As₂O₃.

In one embodiment, the lyopremix or the LCCA has an average particlesize distribution D(90) of from about 2 microns to 10 microns. Statedanother way, the particle size distribution D(90) of the lyopremix asmeasured in microns is 2, 3, 4, 5, 6, 7, 8, 9, and 10 microns or anumber within a range by defined by any two number thereof

In one embodiment, the lyopremix or the LCCA has an average particlesize distribution D(10) of from about 0.2 microns to 3 microns. Statedanother way, the particle size distribution D(10) of the lyopremix asmeasured in microns is 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.9, 1.0, 1.1, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, and 3.0 microns or a number within a range by defined by anytwo number thereof

In one embodiment, the lyopremix or the LCCA has an average particlesize distribution D(50) of from about 0.5 microns to 4 microns. Statedanother way, the particle size distribution D(50) of the lyopremix asmeasured in microns is 0.5, 0.6, 0.7, 0.9, 1.0, 1.1, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0 microns or a numberwithin a range by defined by any two number thereof.

In one embodiment, the lyopremix or the LCCA has a particle size D(90)that is from 10 times smaller than the API and up to 50 times smallerthan the API. Stated another way, the D(90) of the lyopremix is 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, and 50 times smaller than the D(90) of the API.

In one embodiment, the lyopremix or the LCCA surface area is in therange from about from about 0.5 m2/g to about 5 m2/g. In other words,the surface area can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5.0, m2/g, or a numberwithin a range defined by any two numbers herein.

In one embodiment, the lyopremix's or the LCCA's surface area is from 2times to 80 times more than the surface area of the API powder asmeasured by the BET method. Stated another way, the BET surface area ofthe lyopremix is 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, and 80 times the surface area of the APIpowder.

In one embodiment the lyopremix or the LCCA is soluble in cold water. Inanother embodiment, the lyopremix or the LCCA is from about 2× to about30× more soluble in cold water (room temperature) than the API powder.Stated another way, the lyopremix or the LCCA is from about 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 × more soluble in cold water (roomtemperature) than the API powder. In one embodiment, the lyopremix orthe LCCA is soluble in alcohol. In another embodiment, the lyopremix orthe LCCA is from about 2× to 30× more soluble in alcohol than the APIpowder. Stated another way, the lyopremix or the LCCA is from about 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 × more soluble in alcohol than the APIpowder.

From the scanning electron micrograph, it is observed that the lyopremixor the LCCA particles have porous character, which may contribute to thehigher solubility, higher surface area, and the smaller average particlesize of the lyopremix.

In one embodiment of the above pharmaceutical composition, thealkalizing agent comprises sodium hydroxide (NaOH) or sodium carbonate(Na₂CO₃). In another embodiment, the amount of said alkalizing agentadded is about 10% to about 100% the amount of the As₂O₃ powder. In yetanother embodiment, said acid comprises hydrochloric acid (HCl). In oneembodiment, said HCl is about 6 M HCl. In one embodiment, the acid isadded to said vessel to adjust the pH to about 7.2. In one embodiment,the water is added to said vessel after step h)i), h)ii), h)iii), and/orh)iv).

In one embodiment of the invention, the surfactant comprises sodiumlauryl sulfate; Tween 80; betacyclodextrin; poloxamer; tocopherylpolyethylene glycol succinate (TPGS). In one embodiment of theinvention, the surfactant is added to about 0.5% v/v to about 4.0% v/v,but does not exceed about 50% As₂O₃ concentration.

In one embodiment of the invention, the step of lyophilizing comprisesfreezing said As₂O₃ solution to generate a frozen As₂O₃ product anddrying said frozen As₂O₃ product to generate said lyophilized As₂O₃. Inanother embodiment, the freezing step comprises freezing said As₂O₃solution at about −40° C. In another embodiment, the As₂O₃ solution isfrozen at about −40° C. for at least about 6 hours. In one embodiment,the drying step comprises applying heat and a vacuum to said As₂O₃solid. For example, the drying step comprises heating said As₂O₃ productat about −30° C. and about 800 millitorrs for about 60 minutes; heatingsaid As₂O₃ product at −20° C. and 500 millitorrs for about 120 minutes;heating said As₂O₃ product at about −5° C. and about 500 millitorrs forabout 120 minutes; heating said As₂O₃ product at about 10° C. and about500 millitorrs for about 60 minutes; heating said As₂O₃ product at about25° C. and about 500 millitorrs for about 180 minutes. In anotherembodiment, said drying step comprises heating said As₂O₃ product toabout −30° C. and about 800 millitorrs over about 60 minutes and holdingat about −30° C. and about 800 millitorrs for about 60 minutes; heatingsaid As₂O₃ product to about −20° C. and about 500 millitorrs over about60 minutes and holding at about −20° C. and about 500 millitorrs forabout 120 minutes; heating said As₂O₃ product to about −5° C. and about500 millitorrs over about 300 minutes and holding at about −5° C. andabout 500 millitorrs for about 120 minutes; heating said As₂O₃ productto about 10° C. and about 500 millitorrs over about 120 minutes andholding at about 10° C. and about 500 millitorrs for about 60 minutes;and heating said As₂O₃ product to about 25° C. and about 500 millitorrsover about 60 minutes and holding at about 25° C. and about 500millitorrs for about 180 minutes.

In one embodiment, the drying step further comprises, following saidheating of said As₂O₃ product at about 25° C. and about 500 millitorrsfor about 180 minutes, heating said As₂O₃ product at about 25° C. andabout 500 millitorrs for about 120 minutes.

In one embodiment of the above invention, the bulking agent comprisesmannitol. In another embodiment, the one or more lubricants comprisestalc and/or magnesium stearate.

In one embodiment, the pharmaceutical composition above compriseslyophilized arsenic trioxide, mannitol, talc, and magnesium stearate. Inanother embodiment, the pharmaceutical composition of above is acontrolled release oral solid pharmaceutical composition. In anotherembodiment, the pharmaceutical composition above is encapsulated in acapsule.

In one embodiment, the capsule comprises about 1 mg, 5 mg, 10 mg, and 20mg of the pharmaceutical composition above.

This invention also relates to a kit comprising the pharmaceuticalcomposition above and instructions for use of the same.

In one embodiment, this invention relates to a method of treating ahematological malignancy in a patient in need thereof, comprising thestep of administering to the patient a therapeutically effective amountof the pharmaceutical composition described above.

As a matter of example, said hematological malignancy is acute myeloidleukemia, acute nonlymphocytic leukemia, myeloblastic leukemia,promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia,erythroleukemia, myelodysplastic syndrome, acute promyelocytic leukemia,chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cellleukemia, polycythemia vera, Hodgkin's lymphoma, non-Hodgkin's lymphoma,myeloma, giant cell myeloma, indolent myeloma, localized myeloma,multiple myeloma, plasma cell myeloma, sclerosing myeloma, solitarymyeloma, smoldering multiple myeloma, nonsecretary myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, orextramedullary plasmacytoma.

In one embodiment, the hematological malignancy is acute promyelocyticleukemia (APL). In a further embodiment of the invention the APL isnewly diagnosed APL, or a relapsed APL or refractory APL.

In one embodiment, the pharmaceutical composition is administered daily.In another embodiment, the pharmaceutical composition is administereddaily for 52 weeks/year. In yet another embodiment, the pharmaceuticalcomposition is administered in a single dosage range of about 1 mg toabout 50 mg. In another embodiment, the pharmaceutical composition isadministered in a single dosage range of about 0.1 mg/kg body weight toabout 0.3 mg/kg body weight.

In one embodiment of the invention, the patient was previously treatedor is currently being treated with chemotherapy and/or radiation. Inanother embodiment, the method of treatment further comprisesadministering one or more chemotherapeutic agents to said patient. Thechemotherapeutic agent is administered before, after, or simultaneouslywith said pharmaceutical composition. This invention also relates to acomposition comprising LCCA or lyophilized arsenic trioxide, at leastone bulking agent, and at least one lubricant. In one embodiment, thelyophilized arsenic trioxide is produced by a method comprising:

-   -   m. solubilizing As₂O₃ powder in water in a vessel, said        solubilizing comprising, in the following order:        -   ix. adding an alkalizing agent to said vessel to a pH of            about 12 or higher,        -   x. adding an acid to said vessel to adjust the pH to about 7            to about 8,        -   xi. adding a surfactant to said vessel, and        -   xii. and adding water to said vessel to generate an As₂O₃            solution; and lyophilizing said As₂O₃ solution. In one            embodiment, the alkalizing agent comprises sodium hydroxide            (NaOH) or sodium carbonate (Na₂CO₃). In another embodiment            of the invention, the amount of said alkalizing agent added            is about 10% to about 100% the amount of the As₂O₃. In one            embodiment, acid comprises hydrochloric acid (HCl), and            preferably about 6 M HCl. In another embodiment, the acid is            added to said vessel to adjust the pH to about 7.2. In            another embodiment, water is added to said vessel after step            m)i), m)ii), m)iii), and/or m)iv).

In one embodiment, the surfactant comprises sodium lauryl sulfate; Tween80; betacyclodextrin; poloxamer; tocopheryl polyethylene glycolsuccinate (TPGS). The surfactant is added to about 0.5% v/v to about4.0% v/v, but does not exceed about 50% As₂O₃ concentration.

Lyophilization Freezing Step

In one embodiment, the lyophilizing step comprises freezing said As₂O₃solution to generate a frozen As₂O₃ product and drying said frozen As₂O₃product to generate lyophilized As₂O₃. In one embodiment, thelyophilizing step comprises freezing said As₂O₃ solution to at least oneof the temperatures from −50, −49, −48, −47, −46, −45, −44, −43, −42,−41, −40, −39, −38, −37, −36, −35, −34, −33, −32, −31, −30, 29, −28,−27, −26, −25, −24, −23, −22, −21, −20, −19, −18, −17, −16, −15, −14,−13, −12, −11, −10, −09, −08, −07, −06, −05, −04, −03, −02, −01, and 0°C. In another embodiment, the freezing temperature is selected from arange defined by and inclusive of any two numbers above. . In anotherembodiment, the freezing step comprises freezing said As₂O₃ solution atabout −40° C., for at least 6 hours. The freezing step can have severalfreezing sub-steps, progressively decreasing the temperature. Thefreezing step and/or the freezing sub-steps can be from about 5 minutesto about 500 minutes each, that is, including every number in betweenthe range, for example, 6, 7, 8, 9, . . . , 497, 498, 499, and 500minutes.

Sublimation Step

In one embodiment, the sublimation (primary drying) or the secondarydrying takes place at a temperature in the range of −50° C. to 35° C.Stated another way, the sublimation can be effected at the followingtemperatures: −50, −49, −48, −47, −46, −45, −44, −43, −42, −41, −40,−39, −38, −37, −36, −35, −34, −33, −32, −31, −30, −29, −28, −27, −26,−25, −24, −23, −22, −21, −20, −19, −18, −17, −16, −15, −14, −13, −12,−11, −10, −9,−8, −7, −6, −5, −4, −3, −2, −1, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, and 35. In one embodiment, the sublimationtemperature can be any temperature within a range defined by andinclusive of any two numbers above. In one embodiment, more than onesublimation temperatures are used.

The secondary drying can be performed at higher temperatures, forexample, temperatures between 35° C. and 75° C.

The primary or the secondary drying at different temperatures is donefor time in the range of 5 minutes to 500 minutes, that is, includingevery number in between the range, for example, 6, 7, 8, 9, . . . , 497,498, 499, and 500 minutes. Secondary drying can for longer times, forexample, up to 1000 minutes.

Vacuum Application

Vacuum can be applied simultaneously with drying or independently.Vacuum can be applied as the drying continues from one temperature to asecond (higher) temperature, or during holding at one temperature.Vacuum can be applied over multiple steps of drying or only some stepsof drying. Vacuum application can be alternated with drying. The vacuumapplied during the drying step can be in the range of from about 300millitorrs to about 1000 millitorrs. In one embodiment, the vacuum is300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 50, 540, 550, 560, 570,580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850,860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990,and 1000 millitorrs or a vacuum defined by and inclusive of a numberwithin a range defined by any two numbers herein.

The drying step comprises applying heat and a vacuum to said As₂O₃product. In one embodiment, the drying step comprises heating said As₂O₃product at about −30° C. and about 800 millitorrs for about 60 minutes;heating said As₂O₃ product at about −20° C. and about 500 millitorrs forabout 120 minutes; heating said As₂O₃ product at about −5° C. and about500 millitorrs for about 120 minutes; heating said As₂O₃ product atabout 10° C. and about 500 millitorrs for about 60 minutes; and heatingsaid As₂O₃ product at about 25° C. and about 500 millitorrs for about180 minutes. In another embodiment, the drying step comprises heatingsaid As₂O₃ product to about −30° C. and about 800 millitorrs over about60 minutes and holding at about −30° C. and about 800 millitorrs forabout 60 minutes; heating said As₂O₃ product to about −20° C. and about500 millitorrs over about 60 minutes and holding at about −20° C. andabout 500 millitorrs for about 120 minutes; heating said As₂O₃ productto about −5° C. and about 500 millitorrs over about 300 minutes andholding at about −5° C. and about 500 millitorrs for about 120 minutes;heating said As₂O₃ product to about 10° C. and about 500 millitorrs overabout 120 minutes and holding at about 10° C. and about 500 millitorrsfor about 60 minutes; and heating said As₂O₃ product to about 25° C. andabout 500 millitorrs over about 60 minutes and holding at about 25° C.and about 500 millitorrs for about 180 minutes.

In yet another embodiment, following said heating of said As₂O₃ productat about 25° C. and about 500 millitorrs for about 180 minutes, heatingsaid As₂O₃ product at about 25° C. and about 500 millitorrs for about120 minutes.

In one embodiment of the composition, the bulking agent comprisesmannitol, and the one or more lubricants comprises talc and/or magnesiumstearate.

This invention also relates to a composition described above thatcomprises lyophilized arsenic trioxide, mannitol, talc, and magnesiumstearate.

In one embodiment, for the compositions and methods described herein,the LCCA is used in a therapeutically effective amount.

Experimental

The reference product was an injectable As₂O₃ available in injectionform with the strength of 10 mg/10 mL. The development of test productwas provided in solid form for oral use. The test product developmentwas initiated as 1 mg, 5 mg, 10 mg, and 20 mg capsules. Lyophilizationtechnique was chosen to modify the physical and/or chemicalcharacteristics of arsenic trioxide including reducing particle size,increasing surface area, and generating other morphological featuresthat may help its solubility, dissolution of the pharmaceuticalformulation, and/or bioavailability. Suitable excipients were selectedand concentrations were optimized to achieve smooth lyophilizationprocess yielding improvement in the solubility of arsenic trioxide. Thelaboratory-scale stability data for 15 days at 40° C./75% RH were foundsatisfactory.

Reference Product—Injectable Form—Arsenic Trioxide

According to the package insert for the injectable arsenic trioxide,arsenic trioxide causes morphological changes and DNA fragmentationcharacteristic of apoptosis in NB4 human promyelocytic leukemia cells invitro. Arsenic trioxide also causes damage or degradation of the fusionprotein PML/RAR-alpha.

Arsenic trioxide in solution hydrolyzes to its pharmacologically activespecies arsenious acid (AsIII). Monomethylarsonic acid (MMAV), anddimethylarsinic acid (DMAV) are the main pentavalent metabolites formedduring metabolism, in addition to arsenic acid (Asv) a product of AsIIIoxidation.

The pharmacokinetics of arsenical species ([AsIII], [Asv], [MMAV],[DMAV]) were determined in 6 APL patients following once daily doses of0.15 mg/kg for 5 days per week. Over the total single dose range of 7 to32 mg (administered as 0.15 mg/kg), systemic exposure (AUC) appears tobe linear. Peak plasma concentrations of arsenious acid (AsIII), theprimary active arsenical species were reached at the end of infusion (2hours). Plasma concentration of AsIII declined in a biphasic manner witha mean elimination half-life of 10 to 14 hours and is characterized byan initial rapid distribution phase followed by a slower terminalelimination phase. The daily exposure to Asm (mean AUC0-24) was 194ng-hr/mL (n=5) on Day 1 of Cycle 1 and 332 ng-hr/mL (n=6) on Day 25 ofCycle 1, which represents an approximate 2-fold accumulation.

The primary pentavalent metabolites, MMAV and DMAV, are slow to appearin plasma (approximately 10-24 hours after first administration ofarsenic trioxide), but, due to their longer half-life, accumulate moreupon multiple dosing than does AsIII. The mean estimated terminalelimination half-lives of the metabolites MMAV and DMAV are 32 hours and72 hours, respectively. Approximate accumulation ranged from 1.4- to8-fold following multiple dosing as compared to single doseadministration. Asv is present in plasma only at relatively low levels.

Distribution

The volume of distribution (Vss) for AsIII is large (mean 562 L, N=10)indicating that As^(III) is widely distributed throughout body tissues.Vss is also dependent on body weight and increases as body weightincreases.

Metabolism

Much of the As^(III) is distributed to the tissues where it ismethylated to the less cytotoxic metabolites, monomethylarsonic acid(MMA^(V)) and dimethylarsinic acid (DMA^(V)) by methyltransferasesprimarily in the liver. The metabolism of arsenic trioxide also involvesoxidation of As^(III) to As^(v), which may occur in numerous tissues viaenzymatic or nonenzymatic processes. As^(v) is present in plasma only atrelatively low levels following administration of arsenic trioxide.

Excretion

Approximately 15% of the administered arsenic trioxide injection dose isexcreted in the urine as unchanged As^(III). The methylated metabolitesof As^(III) (MMAV, DMAV) are primarily excreted in the urine. The totalclearance of As^(III) is 49 L/h and the renal clearance is 9 L/h.Clearance is not dependent on body weight or dose administered over therange of 7-32 mg.

Developmental As₂O₃

The active pharmaceutical ingredient arsenic trioxide for theseexperiments was purchased from Sigma-Aldrich as white powder, molecularweight of 197.8 g/mole with the general chemical formula of As₂O₃.

For the comparative example, injectable form arsenic trioxide waspurchased as Arsenox® (Manufactured by Naprod Life sciences privatelimited for Intas pharma in India). Arsenic trioxide injection isindicated for induction of remission and consolidation in patients withacute promyelocytic leukemia (APL). It comes in an injection vial formwith strength of 10 mg/10 mL (1 mg/mL) ampule.

Because the reference product was available in the injection form anddevelopment of test product was in solid form for oral use, thereference product was not charged for stability.

No drug excipient study was performed. Solubility studies were conductedas part of development with different surfactants and solvents.

Development of the Formulation Process

Arsenic trioxide, available in injection form, and development gradepowder for oral administration were tested. The powder form of As₂O₃ waslyophilized to modify the physical and/or chemical characteristics ofarsenic trioxide including reducing particle size, increasing surfacearea, and generating other morphological features that may help indissolution and/or bioavailability of the arsenic.

Lyophilization, also known as freeze drying involves removing purifiedwater from a liquid, paste, or solid form, using freezing and vacuumevaporation cycles without melting the ice. Water in solid state (ice)exposed to very low pressure such as vacuum sublimates or passesdirectly from the solid to the gaseous state. Purified water vapor (orother solvent) leaves the capture by freezing with a condenser or coldtrap. This technique retains the volume, appearance and properties ofthe treated product.

Generally, lyophilization cycle comprises the following steps:

-   -   1. Freezing: The product is frozen. This provides a necessary        condition for low temperature drying.    -   2. Vacuum: After freezing, the product is placed under vacuum or        low pressure. This enables the frozen solvent in the product to        vaporize without passing through the liquid phase, a process        known as sublimation.    -   3. Heat: Heat is applied to the frozen product to accelerate        sublimation.    -   4. Condensation: Low-temperature condensation removes the        vaporized solvent from the vacuum chamber by converting it back        to a solid. This completes the separation process.

The first step in the lyophilization process is to freeze a product tosolidify all of its purified water molecules. Once frozen, the productis placed in a vacuum and gradually heated without melting the product.This process, called sublimation, transforms the ice directly intopurified water vapor, without first passing through the liquid state.The purified water vapor given off by the product in the sublimationphase condenses as ice on a collection trap, known as a condenser,within the lyophilizer's vacuum chamber.

Lyophilization has many advantages compared to other drying andpreserving techniques. For example, lyophilization maintains the qualityof the food/biochemical and chemical reagents because they remain attemperatures below the freezing-point during the process of sublimation.Lyophilization is particularly important when processing lacticbacteria, because these products are easily affected by heat.Food/biochemicals and chemical reagents which are lyophilized canusually be stored without refrigeration, which results in a significantreduction of storage and transportation costs. Lyophilization greatlyreduces weight, and this makes the products easier to transport. Forexample, many foods contain as much as 90% purified water. These foodsare ten times lighter after lyophilization. Because they are porous,most freeze-dried products can be easily rehydrated. Lyophilization doesnot significantly reduce volume; therefore purified water quicklyregains its place in the molecular structure of the food/biochemicalsand chemical reagents.

Based on literature and process (different solubility enhancers likesodium lauryl sulfate, Tween 80®, Poloxamer and pH adjusting agents suchas sodium hydroxide, sodium carbonate, sodium alginate, and hydrochloricacid to improve the solubility were selected for initial development.The dissolution medium used was 0.1N HCl, 900 ml, paddle (mixing) at 100rpm for initial developmental activity. Reference product is availablein single strength (10 mg/10 mL) and the test product was initiated fordifferent strengths for oral administration with dose weightproportionality. As reference product was available in 10 mg strength,the formulation development was initiated with 10 mg strength.

Experiment No. L040/1/001

The objective of this experiment was to perform solubility study forarsenic trioxide, using sodium hydroxide as pH adjusting agent,surfactants namely sodium lauryl sulfate and Tween 80®, and differentsolvents such as isopropyl alcohol and ethanol.

In the first step, 4 g of arsenic trioxide was added to 600 mL ofpurified water under stirring conditions in a container. Next, about 35mL of 3 M NaOH (12 g of NaOH dissolved in 100 mL of purified water) wasadded, drop-wise, under magnetic stirring at 1000 rpm for 15 to 30minutes, until a clear solution was formed. To this solution, 1 Lpurified water added. The pH was measured to be 12.7 units. It wasadjusted with a target of 7 to 8 units, using about 16.5 mL of 6 M HCl(105.6 mL added to 200 mL of purified water). A pH of about 7.2 wasobserved. This solution was made up to 2 L with addition of purifiedwater. The pH remained unchanged at 7.3.

TABLE 1 L040/1/001A L040/1/001A No. Ingredients mg/unitquantity/batch 1. Arsenic trioxide 10 4 g 2. 3M NaOH 10.5 35 mL 3. 6MHCl Qs* 16.5 mL 4. Purified water Qs* Up to 2000 mL *Qs—Quantitysufficient

Experiment No. L040/1/001B

The objective of this experiment was to perform solubility study forarsenic trioxide with sodium lauryl sulfate. In the first step, 50 g ofpurified water was taken in glass beaker, to which 0.5 g of sodiumlauryl sulfate was added under stirring until a clear solution wasformed. Next, 5 g of As₂O₃ was added under stirring to this solution.The As₂O₃ did not dissolve in the solution even after a 30-minutesstirring. In the next step, 30 g of purified water was added todetermine if the As₂O₃ would solubilize. No solubility was observed.Even after a further addition of 2 g sodium lauryl sulfate, nosolubility was observed after 30 minutes of stirring. Two batches wereprepared: L040/1/001B1 and L040/1/001B2. The data are reported in Table2 below.

TABLE 2 L040/1/ L040/1/ 001B1 001B2 L040/1/ quantity/ L040/1/ quantity/001B1 batch 001B2 batch No. Ingredients mg/unit (g) mg/unit (g) 1.Arsenic trioxide 10 5 10 5 2. Sodium lauryl sulfate 1 0.5 5 2.5 3.Purified water Qs 80 Qs 80 *Qs—Quantity sufficient

Experiment No. L040/1/001D

The objective of this experiment was to perform solubility enhancementfor arsenic trioxide with cyclodextrin (kleptose). In the first step, 50g of purified water was taken in glass beaker, to which, 0.5 g ofcyclodextrin was added with stirring until clear a solution was formed.To the above solution, 5 g of As₂O₃ drug was added. Even after 30minutes of stirring, As₂O₃ did not dissolve in the solution. Into thissolution, another 30 g of purified water was added. The As₂O₃ drug didnot dissolve. The As₂O₃ drug did not dissolve even after a further 2-gaddition of cyclodextrin followed by a 30-minutes stirring. Table 3summarizes the data for this experiment.

TABLE 3 L040/1/ L040/1/ 001D1 001D2 L040/1/ quantity/ L040/1/ quantity/001D1 batch 001D2 batch No. Ingredients mg/unit (g) mg/unit (g) 1.Arsenic trioxide 10 5 10 5 2. Cyclodextrin 1 0.5 5 2.5 3. Purified waterQs 80 Qs 80 *Qs—Quantity sufficient

Experiment No. L040/1/001E

The objective of this experiment was to perform solubility enhancementfor arsenic trioxide with Tween 80®. In the first step, 50 g of purifiedwater was taken in a glass beaker, to which 0.5 g of Tween 80® was addedunder stirring until clear solution was formed. To this solution, 5 g ofAs₂O₃ drug was added under stirring. The As₂O₃ remained insoluble evenafter 30 minutes of stirring. To this solution, another 30 g of purifiedwater added, but the As₂O₃ remained insoluble. Eve a further addition of2 g of Tween 80® to the solution failed to dissolve the As₂O₃ even after30 minutes of stirring.

TABLE 4 L040/1/ L040/1/ 001E1 001E2 L040/1/ quantity/ L040/1/ quantity/001E1 batch 001E2 batch No. Ingredients mg/unit (g) mg/unit (g) 1.Arsenic trioxide 10 5 10 5 2. Tween 80 ® 1 0.5 5 2.5 3. Purified waterQs 80 Qs 80 *Qs—Quantity sufficient

Experiment No. L040/1/001F

The objective of these experiments was to use various solvents for thesolubility study of As₂O₃.

Isopropyl Alcohol

In a beaker, 1.5 g of As₂O₃ drug was added to 50 mL isopropyl alcohol.The As₂O₃ did not dissolve even after 30 minutes stirring. The drug didnot dissolve even after an additional 30 mL addition of isopropylalcohol ad 30 minutes of stirring.

Ethanol

In a beaker, 1.5 g of As₂O₃ drug was added to 50 mL ethanol. The drugwas insoluble after 30 minutes of stirring. The drug did not dissolveeven after an additional 30 mL of ethanol was added accompanied by 30minutes of stirring.

Thus, from the above series of experiments, it was observed that onlythe L040/1/001A composition formed clear solution.

Experiment No. L040/1/002

The objective of this experiment was to evaluate the exact quantity ofsodium hydroxide required to dissolve the arsenic trioxide and toevaluate the effect of pH on solubility of arsenic trioxide usingdifferent excipients.

Experiment No. L040/1/002A—NaOH Quantity to Solubilize the ArsenicTrioxide

In a glass beaker, 30 mL of purified water was added, and its pH wasmeasured at 6.7 units to 7.0 units. To this solution, 200 mg As₂O₃ drugwas added with stirring. The pH was observed around 5.6. As₂O₃ particleswere observed as floating in the solution. Into this solution was added,in drop-wise fashion, a 1 N NaOH solution (4 g of sodium hydroxidepellets dissolved in 100 mL of purified water). The drug remainedinsoluble even after addition of 0.6 mL of 1N NaOH and 10 minutes ofstirring. The pH was measured around 9.21. A further amount of 1.4 mL ofIN NaOH was added under stirring. The drug was still insoluble, with apH of 12.21. Upon a subsequent addition of 1.6 mL of NaOH, withstirring, the drug was observed to dissolve at a pH of 12.37. Into thissolution was added an extra 200 mg of As₂O₃ drug, which was not found todissolve in the solution (the pH was found to be 12.27). A 2-mL additionof 1N NaOH was undertaken with stirring and the drug was found todissolve at a pH of 12.33. A further 600 mg addition of the drug to thesolution showed the drug to be insoluble at a pH of 12.27. However, when1N NaOH was added gradually (6.9 mL) the drug was found to dissolve at apH 12.39. Thus, 5 mg of sodium hydroxide required to dissolve the 10 mgof arsenic trioxide by keeping 30 ml of purified water as constant.

TABLE 5 Ingredients Quantity mg/unit Arsenic trioxide 1000 mg 10 mg NaOH12.5 mL (500 mg of NaOH) 5 mg Purified water 30 mL Qs* *Qs—Quantitysufficient

Experiment No. L040/1/002B

The objective of this experiment was to evaluate the pH dependentsolubility of arsenic trioxide using L-arginine (pH approx. 4). In abeaker 200 mg of L-arginine was dissolved in 100 mL of purified water (2mg/mL solution) under continuous stirring. From this solution 50 mL wasused to dissolve 50 mg of the As₂O₃ drug. However, no dissolution wasobserved; the pH was noted to be 4.94.

TABLE 6 Ingredients Quantity mg/unit Arsenic trioxide 50 mg 10 mgL-Arginine 100 mg 20 mg Purified water 50 mL Qs* *Qs—Quantity sufficient

Experiment No. L040/1/002D

The objective of this experiment was to evaluate the pH dependentsolubility of arsenic trioxide using sodium bicarbonate (pH approx. of8). In a beaker 200 mg of sodium bicarbonate was dissolved in 100 mL ofpurified water (2 mg/mL solution). From this solution, 50 mL was taken(pH of 8.2) and 50 mg of As₂O₃ drug was added to it. No solubility wasobserved (at a pH of 8.0).

TABLE 7 Ingredients Quantity mg/unit Arsenic trioxide 50 mg 10 mg Sodiumbicarbonate 100 mg 20 mg Purified water 50 mL Qs* *Qs—Quantitysufficient

Experiment No. L040/1/002E

The objective of this experiment was to evaluate the pH dependentsolubility of arsenic trioxide using L-arginine base (pH approx. of 12).In a beaker, 200 mg of L-arginine base was dissolved in 100 mL ofpurified water (2 mg/mL solution) under continuous stirring. From thissolution, 50 mL was used to dissolve 50 mg of the As₂O₃ drug (pH of10.90). However, no dissolution was observed; the pH was noted to be10.53.

TABLE 7.1 Ingredients Quantity mg/unit Arsenic trioxide 50 mg 10 mgL-Arginine 100 mg 20 mg Purified water 50 mL Qs* *Qs—Quantity sufficient

Experiment No. L040/1/002F

The objective of this experiment was to evaluate the pH dependentsolubility of arsenic trioxide using sodium carbonate (pH approx. of12). In a beaker 50 mg of sodium carbonate was added to 50 mL ofpurified water (1 mg/mL solution). To this solution, 50 mg of As₂O₃ drugwas added to it under stirring at a pH of 11.25. No solubility of thedrug was observed. However, when the concentration of sodium carbonatewas gradually increased up to 50 mg of sodium carbonate/mL, the arsenictrioxide was found to be soluble in the solution at a pH of 11.46.

TABLE 8 Ingredients Quantity mg/unit Arsenic trioxide 50 mg 10 mg Sodiumcarbonate 2500 mg 500 mg Purified water 50 mL Qs* *Qs—Quantitysufficient

Thus, overall, the drug was insoluble in entire pH range from 4 to 12when using arginine, arginine base, or sodium bicarbonate, but was foundto be soluble using sodium carbonate with higher concentration (500 mgof sodium carbonate was required for dissolving 10 mg of arsenictrioxide).

Experiment No. L040/1/002G

The objective of this experiment was to repeat the experiment ofL040/1/002A but with bulking agents such as lactose monohydrate andmannitol for subsequent lyophilization. In the first step, 10 g ofarsenic trioxide drug was added to 300 mL purified water under stirring,and then, 125 mL of 1N NaOH added under stirring. The drug was found tobe soluble. This solution was divided into two equal parts:

L040/1/002G1: In the first part, lactose monohydrate was added (3.5 g)under stirring and the solution kept aside for physical observation andthen for lyophilization (pH 12.34). After around 24 hours at roomtemperature, the clear solution turned brown and after 36 hours, theintensity of color increased and turned dark brown, and thus,lyophilization was not carried out.

L040/1/002G2: For the second part, mannitol (3.5 g) was added understirring and the solution was kept aside for physical observation andsubsequent lyophilization (pH 12.64). After around 24 hours, thesolution did not change physically, and the clear solution was chargedfor lyophilization for 24 hours. After lyophilization, the materiallooked very sticky in nature and it was very difficult to fill intocapsules. It is possible that the stickiness was a result of thecorrosive nature of NaOH, or due to a high pH of solution (12.5), orsimply due to the presence of mannitol.

TABLE 8.1 Quantity Ingredients (L040/1/002G) mg/unit Arsenic trioxide 10mg 10 mg NaOH 125 mL 5 mg Purified water 300 mL Qs* Total solution 425mL *Qs—Quantity sufficient

TABLE 8.2 Ingredients L040/1/002G1 L040/1/002G2 Solution (L040/1/002G)212.5 mL 212.5 mL Lactose monohydrate 3.5 g — (Pharmatose 200M) Mannitol(Pearlitol SD 200) — 3.5 g *Qs—Quantity sufficient

In the next step, other surfactants were evaluated as solubilizingagents for arsenic trioxide. Also, one additional experiment wasperformed with sodium carbonate with increased quantity of purifiedwater instead of NaOH. One further experiment also was performed usingNaOH with pH adjustment between 7 and 8. Another experiment wasperformed to evaluate the effect of mannitol on sticky nature afterlyophilization.

Experiment No. L040/1/003

The objective of this experiment was to perform the solubility ofarsenic trioxide using Poloxamer and sodium carbonate.

L040/1/003A: Solubility Enhancement Using Poloxamer

In a beaker, 200 mg of Poloxamer was dissolved in 100 mL of purifiedwater (2 mg/mL solution) under stirring. From this solution, 50 mL wastaken and 50 mg of drug was added. The drug was found to be insoluble. Afurther addition of 50 mg of Poloxamer did not dissolve the drug eithereven after a 20-minute stirring step.

TABLE 9 Quantity Ingredients (L040/1/003A) mg/unit Arsenic trioxide 50mg 10 mg Poloxamer 188 150 mg 30 mg Purified water 50 mL Qs**Qs—Quantity sufficient

L040/1/003B: Solubility Enhancement With Increased Quantity of PurifiedWater

This experiment was similar to L040/1/002F. In a beaker 1250 mg ofsodium carbonate was added to 50 ml (25 mg of sodium carbonate/ml) ofpurified water. To this solution, 50 mg of arsenic trioxide drug wasadded under stirring, but no solubility of the drug was observed (pH of11.25). Adding 100 mL of purified water did not dissolve the drugeither.

TABLE 10 Quantity Ingredients (L040/1/003B) mg/unit Arsenic trioxide 50mg 10 mg Sodium Carbonate 1250 mg 250 mg Purified water 150 mL Qs**Qs—Quantity sufficient

Experiment L040/1/004

The objective of this experiment was to take the trial using sodiumhydroxide and bulking agent (mannitol) and to evaluate the effect of thesticky nature by adjusting the pH around 8 (using 6 M hydrochloric acid)after lyophilization. Arsenic trioxide was dispersed in purified waterand 12 mL of 3 M NaOH was added under stirring, until clear solutionformed. The pH was adjusted using 6 M HCl to between 7 and 8. Purifiedwater was added up to 500 ml to the above solution and it was dividedinto two lots:

-   -   Lot 1: L040/1/004A1: 250 mL loaded for lyophilization for 24        hours.    -   Lot 2: L040/1/004A2: 250 mL, mannitol added under stirring and        loaded for lyophilization for 24 hours.

TABLE 11 Batch Ingredients mg/unit (100 Capsules) Arsenic trioxide 10 mg1 gm NaOH 14.4 mg 12 mL (1.44 g) 6M HCl Qs* Qs* Purified water Qs* 500mL *Qs—Quantity sufficient

TABLE 11.1 L040/1/ L040/1/ L040/1/ L040/1/ 004A1 004A1 004A2 004A2mg/unit g mg/unit G Drug Solution 250 mL 250 mL Pearlitol 100SD 20 1(mannitol) Weight 24.4 1.22 44.4 2.22

Experiment L040/1/004B

The objective of this experiment was to prepare the lyopremix forlyophilization. The trial was taken using sodium hydroxide in solid formwith decreased purified water quantity and using mannitol as bulkingagent. In the first step, arsenic trioxide and NaOH were dispersed inpurified water and stirring was continued until a clear solution wasformed. To this solution, 6 M HCl was added and it was charged forlyophilization for 24 hours. After a 24-hour lyophilization, for theL040/1/004A1 batch, white fluffy powder was observed. For theL040/1/004A2, an off-white color, sticky material was observed, whichwas critical to remove from the petridish. For the L040/1/004B, a whitefluffy powder was observed. It was concluded based on these experimentsthat the sticky nature of the material (lyopremix) is due to themannitol in the solution.

In the first step, the lyophilized form of arsenic trioxide and NaOHwere added to pearlitol SD 200 and sifted through a #40-mesh(400-micron) and blended for 10 min. In the next step, talc andmagnesium stearate were sifted through a #60-mesh (250-micron) and addedto the material from the first step and blended for 5 min. The finalblend was filled into size “0” hard gelatin capsule shells under LaminarAirflow System.

TABLE 12 Composition for Lyopremix L040/1/004B L040/1/004B Batch (500Capsules) Ingredients mg/unit Theoretical Arsenic trioxide 10 5 g NaOH10 5 g 6M HCl Qs* 18 ml Purified water Qs* 80 ml Lyopremix Weight 10 gQs*—Quantity sufficient

The L040/1/004B batch was further processed using mannitol as bulkingagent.

TABLE 12.1 Composition for Final Blend L040/1/004B L040/1/004B Batch(500 Capsules) Ingredients mg/unit Theoretical Lyopremix 20 10 PearlitolSD 200 78 39 Talc 1 0.5 Mg stearate 1 0.5 Total 100

Experiment No. L040/1/005

The objective of this experiment was to evaluate the dissolution ofdrug, as such arsenic trioxide filled in “0” size hard gelatin capsuleshells (which is equivalent to 10 mg). It was found that dissolution wasincomplete even at 60 minutes.

TABLE 13 Dissolution and assay comparison of reference and test productDISSOLUTION (0.1N HCl, 100 rpm, 900 ml, Paddle) Reference Time ProductL040/1/004B L040/1/005 10 min — 67.83 80.31 60 min — 73.46 81.71 Assay110% 94.53 99.71

The next trials were planned using different surfactants in formulationto improve the dissolution.

In the L040/1/006 experiment, the trial taken was similar to L040/1/004Band based on precipitation of solution, solvent (purified water)quantity was optimized as 25 mg/ml of arsenic trioxide. In theL040/1/007, experiment, the trial taken was with and without inclusionof different surfactants to evaluate the dissolution and DifferentialScanning calorimetry (DSC) study. For the arsenic trioxide capsules, theobjective was to prepare drug solution (25 mg/mL of arsenic trioxide)with incorporation of different surfactants.

In the first step, arsenic trioxide was dispersed in 64 mL of purifiedwater. In the next step, 4 g of sodium hydroxide was added to above stepand solubility of drug was observed at pH of 12.82. To this solution, 6M HCl was added and pH was observed to be 8.05. Into this solution,purified water was added and the solution divided into different parts(each part contained 40 ml):

-   -   L040/1/007: 40 ml of drug solution;    -   L040/1/007A: 0.5 g of sodium lauryl sulfate added to 40 ml of        drug solution; and    -   L040/1/007B: 0.5 g of Tween 80® added to 40 ml of drug solution.

The solutions L040/1/007 and L040/1/007A were analyzed by DifferentialScanning calorimetry (DSC) to evaluate the melting point. From the DSCstudies, it is clear that there was no significant change in the meltingpoint.

TABLE 14 Ingredients Qty/batch Drug 4 g NaOH 4 g 6M HCl 16 ml Purifiedwater 144 ml Experiment No. L040/1/007 L040/1/007A L040/1/007B Drugsolution 40 ml 40 ml 40 ml Sodium lauryl 0.5 g sulfate Tween 80 ® 0.5 g

Experiment L040/1/008

The next experiment's objective was to use sodium lauryl sulfate in twodifferent concentrations for lyophilization in a trial similar to theExperiment L040/1/007. Into a beaker, 160 mL of purified water was takenand to this 10 g of arsenic trioxide and 10 g of NaOH were added andstirring was continued until clear solution was formed (pH 12.38). Tothis solution, was added 40 mL of 6 M HCl under stirring (pH of 8.04).Furthermore, 200 mL of purified water was added to above solution understirring (pH 8.18). This solution was then divided into different parts(each part contained 80 mL):

-   -   Part 1: L040/1/008A: As is solution.    -   Part 2: L040/1/008E: 1 g of sodium lauryl sulfate was added to        80 ml of solution under stirring (label claim: 25 mg contains 10        mg of arsenic trioxide).    -   Part 3: L040/1/008F: 0.2 g of sodium lauryl sulfate was added to        80 mL of solution under stirring (label claim: 21 mg contains 10        mg of arsenic trioxide).

Subsequently, all solutions were loaded for lyophilization for 24 hoursand samples were collected after lyophilization.

TABLE 15 L040/1/008 (qty/batch in g) Ingredients L040/1/008A L040/1/008EL040/1/008F Arsenic trioxide 10 mL 10 mL 10 mL NaOH 10 mL 10 mL 10 mL 6MHCl 40 mL 40 mL 40 mL Purified water 400 mL 400 mL 400 mL Drug solution80 mL 80 mL 80 mL Sodium lauryl sulfate — 1.0 0.2 (SLS) Theoreticalyield (g) 4.0 5.0 4.2

TABLE 15.1 Theoretical Label claim Yield Pract. Yield Batch No.(Theoretical) (g) (g) L040/1/008A 20 mg ≈ 10 mg of 4.0 5.4062 (withoutsurfactant) Arsenic trioxide L040/1/008E (SLS) 25 mg ≈ 10 mg of 5.05.7801 Ratio (Drug:surfact): Arsenic trioxide 2:1 L040/1/008F (SLS) 21mg ≈ 10 mg of 4.2 5.0093 Ratio (Drug:surfact): Arsenic trioxide 10:1

The practical yield of each batch was higher than theoretical yield,which was attributed to the moisture uptake by the lyopremix. Based onassay value, the lyopremix was blended with extra-granular material andfilled into capsules.

TABLE 16 Assay and moisture content results: Label claim Moisture AssayBatch No. (Theoretical) content (%) L040/1/008A 20 mg ≈ 10 mg of 12.483.12 (without surfactant) Arsenic trioxide L040/1/008E (SLS): 25 mg ≈10 mg of 11.5 86.13 2:1 Arsenic trioxide L040/1/008F (SLS) 21 mg ≈ 10 mgof 11.8 82.87 10:1 Arsenic trioxide

Based on above assay, the following filling trials were performed.

TABLE 17 Composition for L040/1/009B L040/1/009B Based on Assay BatchActual fill wt. Size Ingredients (mg/unit)) (mg/unit) (g) Lyopremix 2024.060 2.165 (L040/1/008A) Mannitol 78 73.940 6.655 (Pearlitol SD 200)Talc 1 1.000 0.090 Mg stearate 1 1.000 0.090 Total 100 100.000 9.000

TABLE 18 Composition for L040/1/012B L040/1/012B Based on Assay BatchActual fill wt. Size Ingredients (mg/unit) (mg/unit) (g) Lyopremix 2529.020 2.612 Mannitol 75 68.980 6.208 (Pearlitol SD 200) Talc 1 1.0000.090 Mg stearate 1 1.000 0.090 Total 102 100.000 9.000

TABLE 19 COMPOSITION FOR L040/1/013B L040/1/013B Based on Assay BatchActual fill wt size Ingredients (mg/unit) (mg/unit) (g) Lyopremix 21.00025.342 2.281 (L040/1/008F) Mannitol 77.000 72.658 6.539 (Pearlitol SD200) Talc 1.000 1.000 0.09 Mg stearate 1.000 1.000 0.09 Total 100.000100.000 —

The lyophilized form of arsenic trioxide and NaOH (lyopremix) were addedto pearlitol SD 200 and sifted through #40-mesh and blended for 10minutes. Talc and magnesium stearate were sifted through #60-mesh andwere added to above material and blended for 5 minutes. This powder wasfilled into size “2” capsules.

TABLE 20 DISSOLUTION (0.1N HCl, 100 rpm, 900 ml, Paddle) Time in MinutesL040/1/009B L040/1/012B L040/1/013B 15 minutes 73 87 88 60 minutes 75 8990

From the above data, it is clear that a complete dissolution wasobserved in all the batches. In the next step, bigger batch samples,similar to L040/1/012B, and all strengths, 1 mg, 5 mg, 10 mg and 20 mgwere filled. Based on the batch no. L040/1/012B, confirmatory batch(batch no. L040/4/014) was manufactured with all the strengths (1 mg, 5mg, 10 mg, and 20 mg).

TABLE 21 Solution Preparation and Lyophilization Cap. Cap. Cap. Cap.Name of the Ingredient/ Quant. Quant. Quant. Quant. No. Brand NameManufacturer Specification (mg) (mg) (mg) (mg) 1. Arsenic trioxideSigma-Aldrich IH 1 5 10 20 2. Sodium hydroxide J. T. Bakers USP-NF 1 510 20 pellets 3. 6M Hydro-chloric Acid J. T. Bakers USP-NF Qs Qs Qs Qs4. Purified water — Qs Qs Qs Qs 5. Purified water — Qs Qs Qs Qs 6.Sodium lauryl Cognis USP-NF 0.5 2.5 5 10 sulfate (Texapan) Lyopremix(mg) 2.5 12.5 25 50 *Qs—quantity sufficient

TABLE 22 Blending & Lubrication Cap. Cap. Cap. Cap. Name of theIngredient/ Quant. Quant. Quant. Quant. No. Brand Name ManufacturerSpecification (mg) (mg) (mg) (mg) 1. Lyopremix IH 2.5 12.5 25 50 2.Mannitol Roqette USP-NF 7.3 36.5 73 146 (Pearlitol SD 200) 3. Talc(Luzenac) Luzenac USP 0.1 0.5 1 2 4. Magnesium stearate Peter USP-NF 0.10.5 1 2 (Ligamed MF2V) Greven Fill Weight (mg) 10 50 100 200

In the next step, arsenic trioxide and sodium hydroxide pellets weredispersed in 480 ml of purified water and stirring was continued until aclear solution was formed. To this solution was added 120 mL of 6 M HClunder stirring. Sodium lauryl sulfate, 15 g, was added to this solutionunder stirring. An additional 20 mL of purified water was added to abovesolution. This solution was divided into two parts (L040/4/014A andL040/4/014B) and lyophilized for around 30 hours.

TABLE 23 Label claim Batch Numbers (Theoretical) Assay #L040/4/014A 25mg ≈ 10 mg of 82.35 Arsenic trioxide #L040/4/014B 25 mg ≈ 10 mg of 83.35Arsenic trioxide

TABLE 24 L040/4/014B1 mg/unit Qty/batch Ingredients mg/unit (Based onassay) (g) Lyopremix 50 59.98 53.68 (L040/4/014B) Mannitol 146 136.02121.74 (pearlitol SD 200) Talc 2 2 1.79 Magnesium stearate 2 2 1.79Total 200 200 —

In the next step, lyophilized form of arsenic trioxide and NaOH(lyopremix) were added to pearlitol SD 200 and sifted through #40-meshand blended for 10 minutes. Talc and magnesium stearate were siftedthrough #60-mesh and added to above material and blended for 5 minutes.The final blend and suitable capsules and glass vials were prepared asgiven below.

TABLE 25 Strength Batch No Batch size Pack 20 mg L040/4/014B1 400 unitsHDPE 10 mg L040/3/014B1 400 units HDPE 10 mg lubricated L040/3/014B2 350units Glass Vial blend 5 mg L040/2/014B1 400 units HDPE 1 mgL040/1/014B1 400 units HDPE

TABLE 26 Batch No. L040/4/014B1 L040/3/014B1 L040/3/014B2 L040/2/014B1L040/1/014B1 Strength 20 mg 10 mg 10 mg 5 mg 1 mg Capsule Size/VialFilling 2 3 Glass vial 5 5 Fill Weight (mg) 200 100 100 50 10Dissolution (0.1N HCl; 86 92 77 93 100 rpm; 900 mL; Paddle for 15 min.

TABLE 27 Stability Studies-L040/4/014B1 (20 mg) Batch No. L040/4/014B1(20 mg) 40 degree C./75% RH Initial (15 days) Assay 95.15 101.41Moisture content 2.24 2.69 Dissolution (0.1N HCl; 86 98 100 rpm; 900 mL;Paddle at 15 min.)

TABLE 28 Stability Studies-L040/3/014B1 (10 mg) Batch No. L040/3/014B1(10 mg) 40 degree C./75% RH Initial (15 days) Assay 102.4 99.15 Moisturecontent 2.38 2.19 Dissolution (0.1N HCl; 92 100 100 rpm; 900 mL; Paddleat 15 min.)

TABLE 29 Stability Studies-L040/3/014B2 (10 mg lubricated blend) BatchNo. L040/3/014B2 (10 mg lubricated blend) 40 degree C./75% RH Initial(15 days) Assay 98.2 99.78 Moisture content 1.90 2.32

TABLE 30 Stability Studies-L040/2/014B1 (5 mg) Batch No. L040/2/014B1 (5mg) 40 degree C./75% RH Initial (15 days) Assay 98.8 99.95 Moisturecontent 2.63 2.82 Dissolution (0.1N HCl; 77 84 100 rpm; 900 mL; Paddleat 15 min.)

TABLE 31 Stability Studies-L040/1/014B1 (1 mg) Batch No. L040/1/014B1 (1mg) 40 degree C./75% RH Initial (15 days) Assay 94 94.16 Moisturecontent 3.54 3.43 Dissolution (0.1N HCl; 93 92 100 rpm; 900 mL; Paddleat 15 min.)

Pharmacokinetic Analysis in Dogs of Novel Lyophilized FormulationComprising Arsenic and Comparison With Reference Arsenic Trioxide

The purpose of these studies was to determine the oral bioavailabilityand systemic exposure in dogs of a novel formulation comprising arsenicutilizing lyophilization technology. Dog is a well-documented non-rodentspecies used routinely to estimate the pharmacokinetic properties ofnovel agents and formulations in humans.

Study Design

Groups of male dogs (n=3) were administered I.V. formulation of arsenictrioxide (ARSENOX®) at 0.3 mg/kg, unformulated As₂O₃ (SV100) at a doseof 2 mg/dog orally or lyophilized formulation of As₂O₃ (SV101) at a doseof 2 mg/dog, orally. A fourth group of dogs were administered SV101 at 2mg/dog orally for confirmatory analysis at a later date. Blood sampleswere obtained pre-dose and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hpost-dose from the animals in Group 1, and pre-dose and at 0.25, 0.5, 1,2, 4, 6, 8 and 24 h post-dose from the animals in Groups 2, 3 and 4.Blood samples were immediately placed on wet ice and processed to plasmawithin 30 minutes of collection. Plasma samples were analyzed at QPSNetherlands BV for arsenic levels using an ICP-MS method with a lowerlimit of quantification (LLOQ) of 2.50 ng/mL. Individualconcentration-time data were used in the calculation of PK parameters ofarsenic using WinNonlin™ version 5.2.1.

Results

Following IV bolus dosing of ARSENOX®, plasma levels of arsenic reached146 ng/ml at Tmax of 1.3 h and declined with a half-life (t_(1/2)) of10.0 h (FIG. 1 ). Mean systemic clearance was 226 mL/h/kg, and mean Vsswas 2734 mL/kg. Following oral administration, SV100 demonstrated verylimited absorption with an oral bioavailability (F^(b)%) of <10%. Theoral absorption of SV101 (group 3) is nearly identical to ARSENOX®(I.V.) with Cmax of 182±34.5 ng/ml (vs 146±17.6 ng/ml for ARSENOX®) andAUC0-last (h·ng/mL) of 1416±345 (vs 1115±127 for ARSENOX®) indicatingcomplete oral absorption. These data were confirmed by the repeatadministration of SV101 (group 4) which produced nearly identical PKexposure parameters. No tolerability issues were identified during oraladministration of As₂O₃.

CONCLUSION

Animals were dosed with As₂O₃ as intravenous or oral formulation bysingle administration. Study results showed that the absorption rate anddrug exposure of arsenic in the SV101-treated groups were dramaticallyhigher than those in the SV100 group. Complete oral absorption of As₂O₃in the SV101 treated groups was demonstrated by nearly identicalsystemic exposure of arsenic compared to IV administered ARSENOX®.

Characterization of Pharmaceutical Capsules Comprising LyophilizedComposition Comprising Arsenic

The objective of these experiments was to characterize the capsuleformulation comprising arsenic and summarize their physicochemicalproperties. The capsule sizes prepared were 1 mg, 2 mg, 5 mg, 10 mg, and20 mg of the lyopremix formulation comprising arsenic. Four thousandcapsules were prepared. The manufacturing process comprised of (A)Lyophilization, and (B) Blending and Lubrication steps. FIG. 2 shows theprocess flow diagram for capsule making.

(A) Lyophilization

Lyophilization was performed in three stages as follows.

1. Freezing

After solubilization the clear solution was charged into a lyophilizerwith set temperature of −40° C. to freeze the product.

TABLE 32 Freezing Step in Lyophilization Temperature Time Vacuum Stage(° C.) (min) (millitorrs) Freezing −40 60 none Freezing −40 300 noneExtra Freeze −40 5 none (° C.)

2. Primary Drying

This stage is also called the “sublimation” stage. In this stage, dryingwas carried out in presence of vacuum and with increased temperaturegradually up to the material converted into solid form. Processparameters followed for this stage are tabulated below.

TABLE 33 Primary Drying Ramp Hold Temperature Time Vacuum TemperatureTime Vacuum (° C.) (min) (millitorrs) (° C.) (min) (millitorrs) −30 60800 −30 60 800 −20 60 500 −20 120 500 −5 300 500 −5 120 500 10 120 50010 60 500 25 60 500 25 180 500

3. Secondary Drying

The material after primary drying was again dried with optimized vacuumand temperature to remove the bound water.

TABLE 34 Secondary Drying Temperature Time Vacuum (° C.) (min)(millitorrs) 25 120 500

(B) Blending and Lubrication

The assay of the Lyopremix was estimated and the actual quantity of theLyopremix required per capsule was calculated as given below.

Actual quantity of Lyopremix per capsule, in mg, (A)=[(Quantity perCapsule)×100]/[Assay]Quantity of Mannitol to be dispersed per capsule inmg, (B)=Theoretical Quantity of Mannitol×[A−Theoretical Quantity ofLyopremix]Quantity of Mannitol to be dispersed per Batch=B×Batch size

The extra quantity of Lyopremix taken based on the actual assay wascompensated with Mannitol as shown above. Lyopremix and Mannitol werepassed through an ASTM # 40 mesh screen and blended for ten minutes,manually, in a polyethylene bag. This blend was lubricated usingmagnesium stearate and talc, which were pre-sifted through an ASTM # 60mesh for 5 minutes, manually, in a polyethylene bag. This blend was thenfilled into capsules according to the details given below.

TABLE 35 Fill Weight and Capsule Size Fill Weight Size of Strength (mg)Capsule 20 mg 200 1 10 mg 100 3 5 mg 50 4 2 mg 20 4 1 mg 10 4

Physico-Chemical Characterization

Drug product was characterized for its physicochemical properties andcomparative evaluation with raw material was conducted.

1. Particle Size Analysis

The API and the final formulation were characterized for particle sizedistribution by a Malvern Mastersizer. Data are given in the tablebelow.

TABLE 36 Particle Size Distribution Particle Size API Lyopremix FinalFormulation Distribution (micron) (micron) (micron) D(10) 3.9 0.8 1.9D(50) 21.3 2.0 80.4 D(90) 139 4.7 210.4

A significant reduction in PSD at the Lyopremix stage was evident withthe above data which likely helped improve the solubility of the arsenictrioxide in the formulation. Applicants, however, are not wishing to bebound by this theory or for that matter any scientific theory. It shouldbe noted that the PSD of the final formulation also comprises about 75%of other additional excipients.

2. Polymorphism

The final formulation was analyzed for API polymorphic form afterprocessing to evaluate any polymorphic form change from as such API.Even the intermediate stage (Lyopremix) was characterized by XRDdiffractograms. From this study it was concluded that there was no formchange of API in the formulation and it was found to be stable atintermediate stages of manufacturing. Diffractograms are given in FIGS.3-6 , which established that the same polymorphic form was found to bestable throughout the process and in the final formulations.

3. Images Using Scanning Electron Microscope (SEM)

The three samples, that is plain API, Lyopremix, and the Final Blend,were characterized for particle size and morphology characterization bySEM. The images are given in FIG. 7 .

4. FTIR

Characterization of the API, the Lyopremix, and the Final Formulation byFTIR was performed. The spectra of Lyopremix and Final formulation arecomparable to the observed spectrum of the API.

5. Solubility Study

According to the literature, arsenic trioxide is slightly soluble incold water and insoluble in alcohol. So, solubility of the API andLyopremix were studied in these media to evaluate their behavior and theresults are provided below.

TABLE 37 Solubility Studies API Lyopremix Solvent (g/100 g) (g/100 g)Cold water 2.25 40.31 Alcohol 1.86 41.90

The solubility study results indicate that the solubility of the API wasimproved by approximately 20 times by the Lyophilization process of thepresent invention.

6. Chemical Characterization of Drug Product of All Strengths

TABLE 38 Chemical Characterization No. Tests API 1 mg 2 mg 5 mg 10 mg 20mg 1 Description* N/A Complies Complies Complies Complies Complies 2Identification test N/A Complies Complies Complies Complies Complies 3Average wt. of the filled N/A 50.1 60.7 90.2 149.8 275.3 capsules (mg) 4Average wt. of capsule N/A 10.4 20.6 51.3 101.2 201.7 filling (mg) 5Water content by KF, % w/v 2.4 2.5 2.4 2.6 2.3 2.4 Disintegration time(min) N/A 5 min 5 min 6 minutes 6 minutes 5 min 7 Dissolution by ICP-OES10 88 86 95 98 98 (0.1N HCl, Paddle, 100 rpm, 900 mL at 30 min 8 Assayby ICP-OES (%) 97.4 96.0 95.6 97.1 98.5 98.6 *Complies as per therespective specification N/A—not applicable

7. BET Surface Area Measurement

The surface area of the API and the Lyopremix were measured using theBET method for surface area measurement. The specific surface area ofthe arsenic trioxide API was measured at 0.05 m²/g. The specific surfacearea of the lyophilized composition comprising arsenic trioxide, or theLyopremix, was measured at 2.68 m²/g, which is more than a 50-foldincrease in the surface area.

Table 39 below provides the surface area data for the API. FIG. 8provides the BET Surface Area Plot for the API, which shows how the1/[Q(Po/P−1)] depends on Relative Pressure P/Po.

TABLE 39 BET Surface Area API BET Surface Area (API As₂O₃) 0.0479 +/−0.0008 m²/g Slope 116.430262 +/− 1.959314 g/cm³ Conditions STP YIntercept 1.352003 +/− 0.314575 g/cm³ STP C 87.116895 Qm 0.0085 cm³/gSTP Correlation Coefficient  0.9998584 Molecular Cross-Sectional Area0.2100 nm² Relative Pressure Quantity Adsorbed No. (P/Po) (cm³/g STP)1/[Q(Po/P-1)] 1. 0.108368406 0.0087 14.024907 2. 0.154870307 0.009519.275447 3. 0.203970489 0.0102 25.152943

Table 40 below provides the surface area data for the Lyopremix. FIG. 9provides the BET Surface Area Plot for the Lyopremix, which shows howthe 1/[Q(Po/P−1)] depends on Relative Pressure P/Po.

TABLE 40 BET Surface Area Lyopremix BET Surface Area (Lyopremix) 2.68380 +/− 0.0956 m²/g Slope 1.560306 +/− 10.056450 g/cm³ Conditions STP YIntercept 0.061695 +/− 0.012176 g/cm³ STP C 26.290494 Qm 0.6165 cm³/gSTP Correlation Coefficient  0.9993462 Molecular Cross-Sectional Area0.1620 nm² Relative Pressure Quantity Adsorbed No. (P/Po) (cm³/g STP)1/[Q(Po/P-1)] 1. 0.100287631 0.5034 0.221423 2. 0.199710333 0.68030.366816 3. 0.299372148 0.8031 0.532048

The polymorphic studies show that the API remained unchanged during theprocess of manufacturing as well as in the final formulation indicatingits stability. SEM images in FIG. 7 show that the API particles size isreduced drastically during lyophilization. The final blend containsother excipients, and thus, its size looks coarser. The chemicalproperties of the formulation of all the strengths 1, 2, 5, 10, 20 mgcapsules were found satisfactory and complying with the finished productspecification. Significant improvement in dissolution was observed withthe formulation in comparison with API indicating many fold improvementof solubility of arsenic trioxide in the formulation.

What is claimed:
 1. A method for preparing an oral pharmaceuticalformulation comprising a lyophilized composition comprising arsenic orlyophilized arsenic trioxide (As₂O₃), said method comprising: (A)solubilizing As₂O₃ powder in an aqueous medium to form an As₂O₃solution; (B) lyophilizing said As₂O₃ solution to generate a lyopremix;(C) sifting said lyopremix to generate lyophilized As₂O₃ powder; (D)optionally, adding at least one bulking agent to said lyophilized As₂O₃powder; (E) optionally, adding one or more lubricants to saidlyophilized As₂O₃ powder to generate said oral formulation of As₂O₃. 2.A pharmaceutical composition in a solid dosage form suitable for oraladministration, said composition comprising lyophilized compositioncomprising arsenic.
 3. A method of treating a cancer in a patient inneed thereof, comprising the step of administering to the patient atherapeutically effective amount of a pharmaceutical compositioncomprising lyophilized composition comprising arsenic.
 4. Thepharmaceutical composition of claim 2, wherein the LCCA and the bulkingagent constitute about 98% of the pharmaceutical composition (w/w) andthe lubricant comprises about 2% of the pharmaceutical composition(w/w).
 5. The pharmaceutical composition of claim 4, wherein the LCCAconstitutes about 24-29% (w/w) of the pharmaceutical composition and thebulking agent constitutes about 69-74% (w/w) of the pharmaceuticalcomposition.
 6. The pharmaceutical composition of claim 2, wherein thebulking agent is mannitol and/or the lubricant is talc and/or magnesiumstearate.
 7. The pharmaceutical composition of claim 2, wherein thearsenic is arsenic trioxide or a salt thereof.
 8. The pharmaceuticalcomposition of claim 7, wherein the arsenic is arsenic trioxide.
 9. Thepharmaceutical composition of claim 2, wherein the surfactant is sodiumlauryl sulfate (SLS); polysorbate 80; betacyclodextrin; poloxamer; ortocopheryl polyethylene glycol succinate (TPGS).
 10. The pharmaceuticalcomposition of claim 9, wherein the surfactant is SLS or polysorbate 80.11. The pharmaceutical composition of claim 8, wherein the amount of thesurfactant (by weight) does not exceed about 50% the amount of thearsenic trioxide (by weight).
 12. The pharmaceutical composition ofclaim 2, wherein the LCCA comprises lyophilized particles in the D(90)size range from about 2 microns to about 10 microns.
 13. Thepharmaceutical composition of claim 2, wherein the LCCA compriseslyophilized particles having a BET surface area in the range of fromabout 0.5 m²/g to about 5 m²/g.
 14. The pharmaceutical composition ofclaim 2, wherein the solid dosage form is a capsule or tablet.
 15. Thepharmaceutical composition of claim 14, wherein the solid dosage form isa capsule.
 16. The pharmaceutical composition of claim 15, wherein thecapsule contains 0.25 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg,18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48mg, 49 mg, or 50 mg of the LCCA in powder form.
 17. A kit comprising thepharmaceutical composition of claim 2 and instructions for use.
 18. Amethod of treating a cancer in a patient in need thereof, the methodcomprising administering, to the patient, a therapeutically effectiveamount of the pharmaceutical composition of claim
 2. 19. The method ofclaim 18, wherein the cancer is a hematological malignancy.
 20. Themethod of claim 19, wherein the hematological malignancy is an acutemyeloid leukemia (AML); a chronic myeloid leukemia (CIVIL); amyelodysplastic syndrome (MDS); a lymphoma; or multiple myeloma (MM).21. The method of claim 20, wherein the AML is acute promyelocyticleukemia (APL).
 22. The method of claim 21, wherein the APL is newlydiagnosed APL.
 23. The method of claim 21, wherein the APL is relapsedor refractory APL.
 24. The method of claim 18, wherein thepharmaceutical composition is administered once daily.
 25. The method ofclaim 18, wherein the pharmaceutical composition is administered in asingle dosage range of 1 mg to 50 mg.
 26. The method of claim 18,wherein the pharmaceutical composition is administered in a singledosage range of 0.1 mg/kg body weight to 0.3 mg/kg body weight.
 27. Themethod of claim 18, wherein the patient was previously treated or iscurrently being treated with a chemotherapy and/or radiation.